Studies on the mechanism of action of chloramphenicol
by Edward Joseph Morgan
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in Chemistry
Montana State University
© Copyright by Edward Joseph Morgan (1972)
Abstract:
Inhibition of protein synthesis by the antibiotic chloramphenicol has been viewed in the past as
involving inhibition of peptide chain elongation. This view has been supported by chloramphenicol's
inhibition of the puromycin reaction and inhibition only of peptides larger than the tripeptide in the
polyadenylic and" polyuridylic acid directed systems. Evidence is hereby presented to demonstrate that
in a natural mRNA directed in vitro protein synthesizing system, inhibition of protein synthesis by
chloramphenicol is at the level of initiation.
S-30 assisted nascent 14C-labeled material was inhibited by 75% when MS2 mRNA directed protein
synthesis was incubated with a concentration of 2.5 μg./ml. of chloramphenicol, whereas completed in
vitro chains were inhibited by only 25%. Total acid precipitable counts were inhibited by 50%.
Inhibition of nascent material would not be expected to be greater than inhibition of completed chains
if peptide elongation were affected. A model of peptide chain initiation would, however, be expected to
inhibit the nascent material greater than the completed chains.
Tryptic peptide fingerprints were made of both the ribosomal bound and released MS2 mRNA directed
14c-labeled in vitro MS2 coat protein synthesized in the presence of 2.5 μg./ml. of chloramphenicol.
Chloramphenicol was found only to alter the level of completed chains and nascent material, leaving
the distribution and number of tryptic peptides unaffected. This suggests that chloramphenicol's
mechanism of action was neither peptide chain termination, premature release nor random initiation.
Furthermore, since inhibition of peptide chain extension should not have produced a greater inhibition
of nascent material than of completed material, the results suggest inhibition of initiation.
A high salt ribosomal wash, known to contain the recognized bacterial initiation factors, has been
shown to stimulate protein synthesis in the polycytidylic acid directed system. An in vitro MS2 mRNA
directed system was developed which was fully dependent upon addition of these crude initiation
factors. This system utilized DEAE washed ribosomes, crude initiation factors and a fractionated S-100
supernatant, among other things. A concentration of 0.9 μg./ml. of chloramphenicol inhibited total acid
precipitable counts by 50%.
Incorporation of 14C-amino acids independent of crude initiation factors was not inhibited at a
concentration of 10 μg./ml. of chloramphenicol. These results suggest that chloramphenicol's
mechanism of action at low concentrations in a natural messenger RNA directed system is at the level
of initiation, namely at the level of action of the recognized bacterial initiation factors.
/ STUDIES ON THE MECHANISM OF ACTION OF CHLORAMPHENICOL
by
EDWARD J. MORGAN , III
A t h e s i s s u b m itte d to th e G ra d u a te F a c u l t y 'i n p a r t i a l
fu lf illm e n t o f t h e re q u ire m e n ts for th e d e g re e
of
DOCTOR OF PHILOSOPHY
in
C h e m is tr y
Approved:
I)
H e a d , M a jo r D e p a rtm e n t
Ll^dj)rmah, E x a m in ih g " 0 ^ m m itte e
G fa d u a te ^ D e a n •
MONTANA STATE UNIVERSITY
B o ze m a n , M o n ta n a
June, 1 9 7 2
:•
-
•
.*
i ii
A c k n o w le d g m e n ts
"To m ake a g r e a t d ream com e t r u e , th e f i r s t r e q u ir e m e n t i s a
g r e a t c a p a c i t y to d ream ; th e s e c o n d i s p e r s i s t e n c e — a f a ith in th e
d re a m ."
H ans S e ly e , M .D ., P h .D .
The a u th o r th a n k s :
D r. G ordon Ray J u lia n for h is u n w a v e rin g s u p p o rt a n d i n t e l l e c t u a l
g u id a n ce ,
D r. Ray W o lf for c r e a tin g th e o p p o rtu n ity for t h i s s t u d y ,
D r. H e le n C am ero n for h e r a c a d e m ic a s t u t e n e s s a n d b a c t e r i o l o g i c a l
g u id a n c e , and
D r. P a lm e r D . S k a a r for p r o v is io n o f h is g e n e t i c t r a i n i n g .
.j..
:
i v
TABLE OF CONTENTS
Page
V i t a ......................................................................................................................... .
ii
A c k n o w le d g m e n t . , .......................................................
ill
A b s t r a c t ....................... ... : ..............................'............................................. ... .
x
r
1. IN T R O D U C T IO N ............................................................................................
xi
(a) R e c e p to rs a n d d rug a c t i o n . . ’....................... .............................
I
(b) The m o d es o f a c t i o n o f a n tim ic r o b ia l d r u g s .......................
2
(c) A n tim ic ro b ia l d ru g s in p ro te in s y n t h e s i s ..................
3
I n i t i a t i o n ..............................-.................. ................................
I
-
5
E l o n g a t i o n ............. ■.................................. .....................................".
8
T e r m i n a t i o n .........................................................................................
10
(d) C h lo ra m p h e n ic o l in p ro te in s y n t h e s i s ....................................
11
2 . EXPERIMENTAL RATIONALE.................... .......................................... ... .
15
3. MATERIALS AND METHODS ..............................................................., .
19
(a) B a c te ria l a n d b a c te r io p h a g e s t r a i n s ..............................
19
(b) B a c te ria l a n d v i r a l g ro w th m e d ia .......................................
19
(c) Buffers
21
........................................................
(d) R e a g e n ts a n d c h e m i c a ls
..............................
27
(e) R ib o n u c le a s e r e m o v a l ....................... ... ................................. ... .
28
( f) G row th o f p h a g e MS2 on E s c h e r i c h i a c o l i ....................
29
(g) E x tra c tio n o f m e s s e n g e r RNA from p h a g e MS2 . . i . . . .
,32
(h) E x tra c tio n o f p h a g e MS2 c o a t . p r o t e i n .....................................
35
V
™
3 . M a t e r i a l s a n d M e th o d s (c o n tin u e d )
Page
( i ) P r e p a r a tio n o f S - 3 0 e x t r a c t s . .................................................. .
36
( j ) R ib o so m e p r e p a r a t i o n ................................. ... . ...........................
38
(k) DEAE C olum n p r e p a r a t i o n — p u rifie d ' r ib o s o m e s .................
40
( l) P r e p a r a tio n o f pH 5' f r a c t i o n .................... •...............................
40
(m) P r e p a r a tio n o f c ru d e i n i t i a t i o n f a c to r s
.............................. .
41
(n) In v itr o in c o r p o r a tio n p r o c e d u r e s .................... ...... ...................
44
I n c o r p o r a tio n w ith S -3 0 e x t r a c t s
................................. ...
44
C o u n tin g o f t o t a l a c i d p r e c i p i t a b l e c o u n t s .............
46
F in g e rp r in tin g o f th e ^ C in v itr o p e p t i d e s .................
47
I n c o r p o r a tio n w ith p u r if ie d r ib o s o m e s a n d i n i a t i o n f a c t o r s ..........................
54
4 . EXPERIMENTAL RESULTS..........................
(a) In v itr o f in g e r p r in ts
57
..................................................... ...
57
D e v e lo p m e n t o f th e s y s t e m .......................................................
E ffe c t o f Mg
57
c o n c e n t r a t i o n on S -3 0
i n c o r p o r a t i o n s ............. ...
. . . s. ^
K in e tic s o f ^ C - a m i n o a c i d in c o r p o r a tio n
. . . . . .
S tim u la to ry e f f e c t o f MS2 m e s s e n g e r RNA .................
D e p e n d e n c e o f in c o r p o r a tio n on a d d e d
57
57
58
14
C-
a m in o a c i d s ...................
58
E ffe c t o f c h lo r a m p h e n ic o l o n t o t a l a c i d p r e ­
c i p i t a b l e c o u n ts .......................................
.59
vi
4. E x p e rim e n t R e s u lts (c o n tin u e d )
Page
E ffe c t o f c h lo r a m p h e n ic o l on p e p t i d e s r e ­
l e a s e d in v i t r o .........................................................................
60
P e p tid e map o f n a tiv e MS2 c o a t p ro te in '....................
60
P e p tid e m ap o f in v itro MS2 d i r e c t e d r e ­
le a s e d p e p tid e s
..........................................
60
E ffe c t o f c h lo r a m p h e n ic o l o n th e p e p tid e
map o f p e p t i d e s r e l e a s e d in v itro . ..............................
E x p e rim e n ta l r e s u l t s
.......................................
61
61
E ffe c t o f c h lo r a m p h e n ic o l on a c i d p r e c i p i t a b l e c o u n ts o f th e rib o s o m a l bound in
v itro p e p t i d e s
61
P e p tid e map o f th e in v itr o MS2 d ir e c te d
r ib o s o m a l b o u n d p e p t i d e s ........................
62
E ffe c t o f c h lo r a m p h e n ic o l on th e p e p tid e map
o f th e r ib o s o m a l bou n d in v itr o p e p t i d e s
(b) The ro le o f rib o s o m a l w a s h f a c to r s
............. ...
............................. ...
63
63
Mg"+ t i t r a t i o n c u rv e o f th e p u rif ie d in v itro
s y s t e m ................
T itra tio n c u rv e
63
o f c ru d e i n i t i a t i o n fa c to r s
a g a i n s t t h e p u rif ie d s y s t e m ........................................... .
K in e tic s o f
64
14
C -a m in o a c i d in c o r p o r a tio n o f
th e p u r if ie d s y s t e m ................................. . . . ' . . . . . .
65
E ffe c t o f c h lo r a m p h e n ic o l on t o t a l p r e c i p i t a b l e c o u n ts o f th e p u r if ie d s y s t e m .........................
I
65
v ii
4. E x p e rim e n ta l R e s u lts (c o n tin u e d )
Page
E ffe ct o f c ru d e i n i t i a t i o n fa c to rs on p u rif ie d
s y s te m r e - i n c u b a t e d a f t e r a tw o - h o u r
c e n t r i f u g a t i o n ........................................................................
66
D e p e n d e n c e o f th e p u rif ie d s y s te m on th e
pH 5' f r a c t i o n .................................................................. .
67
\
E ffe c t o f c h lo r a m p h e n ic o l on i n i t i a t i o n f a c 14
to r d e p e n d e n t a n d i n d e p e n d e n t C -a m in o
a c i d i n c o r p o r a t i o n ....................................................
68
5. D I S C U S S I O N ...................................................................................................
70
TABLES AND FIG U R E S....................................
80
LITERATURE CITED
...........................' .............. .......................................'. . .
j
s
127
LIST OF TABLES
T a b le
I.
P r o p e r tie s o f th e E c o ll i n i t i a t i o n f a c t o r s ....................................
Page
80
ix
LIST OF FIGURES
Fig u re
Page
I . S c h e m a tic r e p r e s e n t a t i o n o f th e e v e n t s d u rin g p r o ­
t e i n s y n t h e s i s .......................... ...................... ... .............................. ... .
81
2 . S tru c tu re o f c h lo ra m p h e n ic o l .............................. ............................. .
82
3 . Amino a c i d s e q u e n c e o f MS2 v i r u s c o a t p r o t e i n ............. ...
83
4 . UV s p e c tru m o f h ig h ly p u rif ie d MS2 v i r u s ....................................
84
5 . Flow d ia g ra m i l l u s t r a t i n g th e g ro w th a n d p u r if ic a tio n
o f MS2 v i r u s ....................... ... . . .
............................................................85
6. Flow d ia g ra m i l l u s t r a t i n g th e e x t r a c t i o n o f MS2 RNA
a n d c o a t p ro te in from th e b a c te r io p h a g e M S 2
...........................
86
7. D iag ram o f th e d im e n s io n s o f a n MS2 f i n g e r p r i n t ....................
86
8. E ffe c t o f Mg++ c o n c e n tr a tio n on S -3 0 i n c o r p o r a t i o n s .............
88
9 . K in e tic s o f
14
C -a m in o a c i d in c o r p o r a tio n ..............................
10. S tim u la to ry e f f e c t of MS2 m e s s e n g e r R N A .......................
11. D e p e n d e n c e o f in c o r p o r a tio n on a d d e d
.,90
92
14
C -a m in o
a c i d s .....................................................................................
94
12. E ffe c t o f c h lo ra m p h e n ic o l on t o t a l a c i d p r e c i p i t a b l e
c o u n ts
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
96
13. E ffe c t o f c h lo ra m p h e n ic o l on p e p t i d e s r e l e a s e d in
v itr o
........................................................
14. P e p tid e map o f n a tiv e MS2 c o a t p r o t e i n ........................................
98
100
15. P e p tid e map o f th e in v itro MS2 d i r e c t e d r e l e a s e d
p e p t i d e s ....................................
102
X
F ig u re
'
Page
16. E ffe c t o f c h lo ra m p h e n ic o l on th e p e p t i d e map o f p e p ­
t i d e s r e l e a s e d in v i t r o ................................. . .....................................
104
17. E ffe ct o f c h lo ra m p h e n ic o l on a c i d p r e c i p i t a b l e c o u n ts
o f th e rib o s o m a l b o u n d in v itro p e p t i d e s
....................................
106
18. P e p tid e map o f th e in v itr o MS2 d i r e c t e d rib o s o m a l
b o u n d p e p t i d e s ..................................................................... ......................
108
19. E ffe c t o f c h lo r a m p h e n ic o l on th e p e p t i d e map o f th e
rib o s o m a l b o u n d in v itr o p e p t i d e s .....................................................
HO
2 0 . Q u a n tita tiv e e f f e c t o f c h lo ra m p h e n ic o l on th e r i b o ­
s o m a l b o u n d m v itro tr y p t ic p e p t i d e s
................................. ...
2 1 . Mg++ t i t r a t i o n c u rv e o f th e p u r if ie d in v itro s y s te m
. . . . .
112
114
2 2 . T itra tio n c u rv e o f c ru d e i n i t i a t i o n fa c to r s a g a i n s t th e
p u rif ie d s y s te m
........................................................... ...
116
2 3 . K in e tic s o f ^ C - a m i n o a c id in c o r p o r a tio n o f th e p u r i­
fie d s y s t e m ...................................................................................................
118
2 4 . E ffe c t o f c h lo ra m p h e n ic o l on t o t a l a c i d p r e c i p i t a b l e
c o u n ts o f th e p u rif ie d s y s t e m ............. .......................................... .
120
2 5 . E ffe c t o f c ru d e i n i t i a t i o n f a c to r s on a p u r if ie d s y s t e m
r e - i n c u b a t e d a f te r a 2 - h o u r c e n tr if u g a tio n ...........................
122
2 6 . D e p e n d e n c e o f th e p u r if ie d s y s t e m on th e pH 5'
fr a c tio n
................................................. ..........................................
124.
2 7 . E ffe c t o f c h lo ra m p h e n ic o l on i n i t i a t i o n fa c to r d e p e n d ­
e n t a n d in d e p e n d e n t "^C -am in o a c i d i n c o r p o r a t i o n .................- 126
xi
ABSTRACT
I n h ib i tio n o f p r o te in s y n t h e s i s b y th e a n t i b i o t i c c h lo ra m p h e n ic o l
h a s b e e n v ie w e d in th e p a s t a s in v o lv in g in h ib iti o n o f p e p t i d e c h a in
e l o n g a t i o n . This v i e w h a s b e e n s u p p o r te d b y c h l o r a m p h e n i c o l 's i n h i b i ­
tio n o f th e p u ro m y c in ■r e a c t i o n a n d i n h ib iti o n o n ly o f p e p t i d e s la r g e r
th a n th e tr i p e p t i d e in th e p o l y a d e n y l i c and" p o ly u r i d y lic a c i d d i r e c t e d
s y s t e m s . E v id e n c e is h e r e b y p r e s e n t e d to d e m o n s tr a te t h a t in a n a tu r a l
mRNA d i r e c t e d in v itro p ro te in s y n t h e s i z i n g s y s t e m , i n h ib iti o n o f p ro ­
t e i n s y n t h e s i s by c h lo r a m p h e n ic o l i s a t t h e l e v e l o f i n i t i a t i o n .
8 - 3 0 a s s i s t e d n a s c e n t ^ C - I a b e l e d m a te r ia l w a s i n h i b i t e d by 75%
w h e n MS2 mRNA d i r e c t e d p r o te in s y n t h e s i s w a s in c u b a t e d w ith a c o n ­
c e n t r a t i o n o f 2.5 p g ./m l . o f c h l o r a m p h e n i c o l , w h e r e a s c o m p le te d in v itro
c h a i n s w e re i n h i b i t e d b y o n ly 25%. T o ta l a c i d p r e c i p i t a b l e c o u n ts w e re
i n h i b i t e d by 50%. I n h ib i tio n o f n a s c e n t m a te r ia l w o u ld n o t b e e x p e c t e d
to b e g r e a t e r th a n i n h ib iti o n o f c o m p le te d c h a in s i f p e p t i d e e lo n g a tio n
w e re a f f e c t e d . A m o d el o f p e p tid e c h a in i n i t i a t i o n w o u ld , h o w e v e r , be
e x p e c t e d to i n h i b i t th e n a s c e n t m a te r ia l g r e a t e r th a n th e c o m p le te d
c h a in s.
T ry p tic p e p tid e f in g e r p r in ts w e re m ad e o f b o th th e rib o s o m a l
b o u n d a n d r e l e a s e d MS2 mRNA d i r e c t e d 1 4 c - l a b e l e d in v itr o MS2 c o a t
p r o te in s y n t h e s i z e d in th e p r e s e n c e o f 2.5 p g ./m l. o f c h lo r a m p h e n i c o l.
C h lo ra m p h e n ic o l w a s found o n ly to a l t e r th e l e v e l o f c o m p le te d c h a in s
a n d n a s c e n t m a t e r i a l , l e a v i n g th e d i s t r i b u t i o n a n d n u m b e r o f tr y p tic
p e p t i d e s u n a f f e c t e d . This s u g g e s t s t h a t c h l o r a m p h e n i c o l 's m e c h a n is m
o f a c t i o n w a s n e i t h e r p e p t i d e c h a in t e r m i n a t i o n , p re m a tu re r e l e a s e n o r
random i n i t i a t i o n . F u r th e r m o r e , s i n c e in h ib iti o n o f p e p t i d e c h a in e x t e n ­
s io n s h o u ld n o t h a v e p ro d u c e d a g r e a t e r i n h ib iti o n o f n a s c e n t m a te r ia l
t h a n o f c o m p le te d m a t e r i a l , th e r e s u l t s s u g g e s t i n h ib iti o n o f i n i t i a t i o n .
A h ig h s a l t r ib o s o m a l w a s h , k n o w n to c o n ta i n th e r e c o g n i z e d b a c ­
t e r i a l i n i t i a t i o n f a c t o r s , h a s b e e n sh o w n to s t i m u l a t e p r o te in s y n t h e s i s
i n th e p o l y c y t i d y l i c a c i d d i r e c t e d s y s t e m . An in v itro MS2 mRNA
d i r e c t e d s y s t e m w a s d e v e lo p e d w h ic h w a s fu lly d e p e n d e n t upon a d d i ­
tio n o f t h e s e c ru d e i n i t i a t i o n f a c t o r s . T h is s y s te m u t i l i z e d DEAE
w a s h e d r i b o s o m e s , c ru d e i n i t i a t i o n f a c t o r s a n d a f r a c t i o n a t e d S-100
s u p e r n a t a n t , am ong o th e r t h i n g s . A c o n c e n tr a tio n o f 0.9 [Ug./ml. of
c h lo r a m p h e n ic o l i n h i b i t e d t o t a l a c i d p r e c i p i t a b l e c o u n ts b y 50%.
I n c o r p o r a tio n o f ^ C - a m i n o a c i d s in d e p e n d e n t o f c ru d e i n i t i a t i o n
f a c to r s w a s n o t i n h i b i t e d a t a c o n c e n t r a t i o n o f 10 p g ./m l. o f c h lo ra m ­
p h e n i c o l . T h e s e r e s u l t s s u g g e s t t h a t c h l o r a m p h e n i c o l 's m e c h a n is m of
a c t i o n a t lo w c o n c e n t r a t i o n s in a n a t u r a l m e s s e n g e r RNA d i r e c t e d s y s ­
tem i s a t t h e l e v e l o f i n i t i a t i o n , n a m e ly a t th e l e v e l o f a c t i o n o f th e
re c o g n ize d b a c te ria l in itia tio n f a c to r s .
/
I.
INTRODUCTION
(a) R e c e p to rs a n d drug a c ti o n
The m o s t f u n d a m e n ta l a s p e c t o f p h a rm a c o d y n a m ic s i s th a t w h ic h
d e a l s w ith th e m e c h a n is m o f drug a c t i o n .
The k n o w le d g e o f m o le c u la r
m e c h a n is m s o f a n tim ic r o b ia l d ru g s h a s i n c r e a s e d e x p o n e n t i a l l y during
th e p a s t s e v e r a l y e a r s . This a d v a n c e h a s p a r a l l e l e d , a n d w a s in la rg e
p a r t a b y - p r o d u c t of, i n c r e a s e d u n d e r s ta n d in g o f th e r e p l i c a t i o n o f g e n ­
e t i c in fo rm a tio n a n d i t s t r a n s l a t i o n in to th e s y n t h e s i s o f s p e c i f i c
p ro te in s.
As e a r ly a s 1 8 6 8 , e v e n b e fo re L a n g le y c o in e d th e term " r e c e p t i v e
s u b s t a n c e " , h e s u g g e s t e d t h a t d r u g / c e l l i n t e r a c t i o n s , a n d h e n c e th e
a c t i o n s a n d e f f e c t s o f d r u g s , w e re p r o b a b ly g o v e rn e d b y t h e la w o f m a s s
a c t i o n . This v ie w w a s e x t e n s i v e l y d e v e lo p e d b y A. J. C la rk in th e
1 9 2 0 ' s , a n d i t re m a in s th e k e y s to n e o f m o s t t h e o r i e s o f drug a c t i o n .
C o n s e q u e n t l y , t h e o r i e s o f drug a c t i o n a re q u ite s im ila r to t h e o r i e s o f
e n z y m e a c t i o n a n d are e s s e n t i a l l y i d e n t i c a l w h e n a drug s e r v e s a s an
a n t i m e t a b o l i t e or e n z y m e i n h i b i t o r . The c e l l u l a r c o m p o n e n t d ir e c tly i n ­
v o lv e d in th e i n i t i a l a c t i o n o f a drug i s u s u a l l y te rm e d i t s r e c e p to r .
R e c e p to r s i t e s a re s i m i l a r to th e a c t i v e s i t e s o f e n z y m e s . C a r b o x y l,
a m in o , s u l f h y d r y l , p h o s p h a t e a n d s i m i l a r r e a c t i v e g ro u p s a re th o u g h t to
be u t i l i z e d in a s p a t i a l l y o r ie n te d p a t t e r n c o m p le m e n ta ry to t h a t o f th e
d ru g s w ith w h ic h t h e y r e a c t . The b in d in g o f drug to r e c e p t o r i s th o u g h t
2
to be a c c o m p lis h e d m a in ly b y i o n ic a n d o th e r r a th e r w e a k v a n d e r W a a ls
ty p e f o r c e s , a lth o u g h o c c a s i o n a l l y a firm c o v a l e n t bond i s fo r m e d .
(b) The m o d e s o f a c t i o n o f a n tim ic r o b ia l d ru g s
A n tim ic ro b ia l a g e n t s m ay be c o n v e n ie n tly g ro u p e d a c c o r d in g to
t h e i r m o le c u la r m e c h a n is m o f a c t i o n .
1.
A gents t h a t im p e d e r e p l i c a t i o n o f g e n e t i c m a t e r i a l . N a lid ix ic
a c i d f a lls w ith in t h i s c a te g o r y a n d i s a n e f f e c t i v e a g e n t a g a i n s t b a c ­
t e r i a , p rim a rily th o s e w h ic h a re Gram n e g a t i v e . It i s s t r u c t u r a l l y r e ­
l a t e d to th e p u r i n e s , a n d h a s b e e n sh o w n to b lo c k DNA s y n t h e s i s in
s u s c e p t i b l e o rg a n is m s ( M c N a ll, 19 60).
2.
A g en ts t h a t im p a ir t r a n s l a t i o n o f g e n e t i c in f o r m a tio n . Such
d ru g s in h i b i t e i t h e r p ro te in s y n t h e s i s (th e t e t r a c y c l i n e s a n d e ry th ro m y ­
cin) or in d u c e fo rm atio n of d e f e c t i v e p r o te in m o le c u le s (k a n a m y c in a n d
n e o m y c in ) . The form er a re b a c t e r i o s t a t i c a n d th e l a t t e r b a c t e r i o c i d a l .
3 . A g en ts t h a t im p a ir t r a n s c r i p t i o n o f g e n e t i c m a t e r i a l . Purom yc in i s a d rug o f th is c l a s s .
The drug s t r u c t u r a l l y r e s e m b l e s t r a n s f e r
RNA, a n d forms a p e p tid e b o n d w ith th e g ro w in g p e p tid e c h a i n c a u s in g
p re m a tu re r e l e a s e o f th e g ro w in g c h a i n .
4. A gents t h a t a l t e r s tr u c tu r e a n d fu n c tio n of th e c e l l w a l l . The
p e n i c i l l i n s a re m o s t r e p r e s e n t a t i v e o f t h i s g r o u p , i n h ib iti n g c r o s s l i n k ­
in g (tra n s p 'e p tid a tio n ) o f th e g l y c o p e p t id e c e l l w a ll (T ip p er &
S c h ro m in g e r, 1965).
3
V
5.
A g en ts t h a t r e s t r i c t f u n c tio n o f th e c e l l m e m b r a n e . Polym yxin B
a n d c o l i s t i n (P olym yxin E) a re d ru g s o f t h i s c l a s s .
Both a c t a s c a t i o n i c
d e t e r g e n t s w ith a n a f f in it y for p h o s p h a t e r a d i c a l s , t h e r e b y a lte r in g th e
o s m o t i c b a r r ie r f u n c tio n o f th e c e l l m em b ran e (N e w to n , 1 9 5 4 ).
For th e m o le c u la r b i o l o g i s t , d ru g s t h a t i n h ib it th e t r a n s c r i p t i o n
a n d t r a n s l a t i o n o f g e n e t i c in fo rm a tio n h a v e b e e n v i t a l t o o l s in th e s t e p ­
w i s e d i s s e c t i o n o f th e m e c h a n is m s o f g e n e t i c e x p r e s s i o n .
One of th e s e
d ru g s , c h l o r a m p h e n c o l ,. is co m m o n ly u s e d a s a n i n h ib ito r o f p ro te in
b i o s y n t h e s i s . A lth o u g h d i s c o v e r e d in 1 9 4 3 , i t s m o le c u la r m e c h a n is m o f
a c t i o n h a s re m a in e d o b s c u r e .
In c l i n i c a l u s e , t h e s e s a m e in h ib ito r s of g e n e a c t i o n h a v e b e e n
p o t e n t w e a p o n s a g a i n s t m ic ro b ia l i n f e c t i o n s a n d n e o p l a s t i c p r o c e s s e s .
A s s o c i a t e d w ith t h e p o t e n c y o f t h e s e d ru g s a re s e r io u s a n d lif e t h r e a t ­
e n in g s i d e e f f e c t s .
The i a t r o g e n i c l e u k e m i a s o f a n t i n e o p l a s t i c a g e n ts
a n d c h lo r a m p h e n ic o l in d u c e d a p l a s t i c a n e m ia s a re a l l to o common in
c lin c ia l p ra c tice .
(c) A n tim ic ro b ia l d ru g s in p ro te in s y n t h e s i s
The am o u n t o f k n o w le d g e c o n c e r n in g p ro te in b i o s y n t h e s i s h a s e x ­
p lo d e d in t o a v a s t s e r i e s o f d e l i c a t e r e a c t i o n s a n d s u b r e a c t i o n s s in c e
t h e n o tio n in th e e a r ly 1 9 4 0 's b y B ra c h e t a n d C a s p e r s o n t h a t RNA m ight
b e i n v o lv e d in p r o te in s y n t h e s i s (B r a c h e t , 1 9 4 2 ). R e q u ire m e n ts for s p e ­
c i f i c RNA s p e c i e s , r i b o s o m e s , s u p e r n a t a n t f a c t o r s , r ib o s o m a l fa c to r s
\
4
a n d e n e r g y s o u r c e s h a v e b e e n d e m o n s t r a t e d , a n d fu rth e r re f in e m e n ts o f
t h e s e m e c h a n is m s a r e c e r t a i n to e m e r g e . •
It i s a s i m p l i s t i c n o tio n to a s s u m e t h a t a p a r t i c u l a r a g e n t in h ib its
p r o te in s y n t h e s i s a t " i n i t i a t i o n " ; " e l o n g a t i o n " , or " te r m in a tio n " . The
d e f i n i t i o n s o f t h e s e s t e p s a s w e ll a s th e a c t u a l m o le c u la r m e c h a n is m s
o v e r l a p , a n d l i t t l e i s s a i d o f th e d e t a i l e d in h ib ito r y r e a c t i o n i t s e l f .
I n d e e d , w h a t w a s .c o n s id e re d i n i t i a t i o n in 1970 w a s sh o w n to be a p a rt
o f e lo n g a t io n in 1971 (T h ach & T h a c h , 1 9 7 1 ). A c c o rd in g ly , a d e t a i l e d
r e v ie w o f p r o te in b i o s y n t h e s i s i s in o r d e r .
P ro te in s y n t h e s i s m ay b e c o n v e n i e n t l y c a t e g o r i z e d in to th re e
g ro u p s o f p r o c e s s e s a s d e f in e d below :
1.
I n i t i a t i o n . The a s s e m b l y o f t h e m o le c u la r b i o l o g i c a l s u b u n its
up to b u t n o t in c lu d in g t r a n s l o c a t i o n o r fo rm atio n o f t h e f i r s t p e p tid e
bond.
2.
E lo n g atio n .
The fo rm atio n o f th e f i r s t p e p tid e b o n d , a n d a ll
p e p tid e b o n d s t h e r e a f t e r , a s d i r e c t e d b y a s p e c i f i c m e s s e n g e r KNA
(mRNA).
'3 .
T e rm in a tio n a n d r e l e a s e . The s t e p s - t a k i n g p la g e a f te r th e
c o m p le tio n of th e f in a l p e p tid e b o n d , r e s u l t i n g in a p o ly p e p tid e c h a in
r e l e a s e d from th e p r o te in s y n t h e s i z i n g s u b u n i t s .
It w a s w e ll e s t a b l i s h e d b y 1963 ( W a ts o n , 19 63) t h a t p ro te in b i o ­
s y n t h e s i s u t i l i z e d th e g e n e t i c m e s s a g e c o d e d by mRNA, a n d w ith th e
h e lp o f s p e c i f i c t r a n s f e r RNA1s , th e mRNA formed a c o m p le x w ith
5
rib o s o m e s a llo w in g th e g e n e t i c m e s s a g e to be t r a n s l a t e d .
S in c e t h e n ,
th e in d iv id u a l s t e p s w ith in th is p r o c e s s h a v e b e e n ex am in ed ., a n d fu r­
th e r r e q u ire m e n ts h a v e b e e n s h o w n .
I n it ia ti o n
I n i t i a t i o n i s d e f in e d a s t h o s e s t e p s o c c u r rin g up t o , b u t n o t i n ­
c lu d in g th e fo rm atio n o f th e f i r s t p e p t i d e b o n d . F u r th e r m o r e , i n i t i a t i o n
d o e s n o t i n c lu d e a n y m o v e m e n t o f th e rib o s o m e r e l a t i v e to th e mRNA
(T h ac h & T h a c h , 1971).
P ro te in s y n t h e s i s o c c u r s in th e form o f an a c i d p r e c i p i t a b l e p o l y ­
p e p tid e w h e n a m ix tu re of r ib o s o m e s w a s h e d w ith a h ig h s a l t b u f f e r , a
s y n t h e t i c mRNA-, GTP a s an e n e r g y s o u r c e , s p e c i f i c d i v a l e n t c a t i o n s ,
s u p e r n a ta n t, f a c t o r s , a n d th e a p p r o p r ia te
14
C am ino a c i d s a re i n c u b a t e d
a t 3 7 ° C ( N a t h a n s , N o t a n i , S c h w a rz & Z i n d e r , 1962). S u ch a s y s te m w ill
n o t , h o w e v e r , a llo w for t r a n s l a t i o n o f n a t u r a l mRNA's u n l e s s fa c to rs
r e c o v e r e d in th e h ig h s a l t rib o s o m a l w a s h a re a d d e d (B raw erm an &
E i s e n s t a d t , 1966; S t a n l e y , S a la s , W a h b a & O c h o a , 1 9 6 6 ). The r i b o ­
s o m a l w a s h c o n ta i n s i n i t i a t i o n f a c to r s a s d e fin e d b y t h e i r ro le in th e
i n i t i a t i o n o f p ro te in s y n t h e s i s . They h a v e b e e n e x t e n s i v e l y p u rifie d
(R e v e l, b e l o n g , Brawerm an & G r o s , 19 68; R e v e l, H e r z b e r g , B e c a re v ic &
G ro s s, 1968),, a n d a sum m ary o f t h e i r p r o p e r tie s a p p e a r s in T a b le I.
In o rd e r to a d e q u a te ly d is c u s s th e ro le o f in itia tio n fa c to rs in
p ro te in s y n t h e s i s , i t is h e lp fu l to re fe r to a v is u a l m o d e l. I t is
6
r e c o g n i z e d t h a t s u c h a m o d el s u f f e r s from a l l th e l i m i t a t i o n s o f a n y
a tte m p t to g r a p h i c a l l y d i s p l a y a c o m p le x b io l o g i c a l p r o c e s s (F ig . I).
The f i r s t s t e p in i n i t i a t i o n i s th e fo rm atio n o f c o m p le x (I), co m ­
p r i s e d o f a 30s r ib o s o m a l s u b u n i t , f-m e t-tR N A , i n i t i a t i o n fa c to r s fj a n d
Tg / a n a p p r o p r ia te i n i t i a t i o n c o d o n (in th is c a s e AUG), G T P , a n d the
r e q u i s i t e d i v a l e n t c a t i o n s (T h ach & T h a c h , 1971).
A lthough O h t a , S a rk a r & T h a c h (1967) a n d L e n g y e l & So il (19 69)
h a v e a rg u e d t h a t f-m e t-tR N A b in d s to t h e "A" ( a c c e p to r) s i t e , T h ach &
T h a c h (1971) h a v e a rg u e d t h a t th e u ltim a te f u n c tio n a l b in d in g
o f the
fo rm er i s to an " u n a c c o m m o d a te d P" s i t e , or more s u c c i n c t l y , to th e
" p re P" s i t e (P for p e p tid y l), a lth o u g h i t m ay h a v e p a s s e d th ro u g h an
"A" s i t e .
The " p re P" s i t e i s th e s a m e a s th e "P" s i t e , h o w e v e r th e
a m in o a c i d p o rtio n o f th e aa-tR N A c o m p le x i s n o t o r i e n t a t e d in a s p a ­
t i a l l y c o r r e c t m a n n e r to t h e p e p tid y l t r a n s f e r a s e . W h e n 't h e tRNA i s in
th e "P" s i t e , i t i s c o m p le x (I I I ) . R efer to F ig . I.
O n c e c o m p le x (I) i s fo rm e d , a 5 0 s r ib o s o m a l s u b u n i t j o i n s to form
c o m p le x ( I I ) , a r a t h e r s h o r t - l i v e d in t e r m e d i a t e (T hach & T h a c h , 1971).
U pon t h e fo rm a tio n o f c o m p le x (II), fj. d i s s o c i a t e s to r e c y c l e a g a i n .
C o m p le x (II) im m e d ia te ly p r o c e e d s to c o m p le x (III), d u rin g w h ic h tim e
t h e f-m e t-tR N A i s " a c c o m m o d a te d " ( s t e r i c a l l y s h ifte d ) in to th e "P" s i t e
from th e " p re P" s i t e .
GTP i s h y d ro ly z e d a s th e e n e r g y s o u r c e for t h i s
s t e p a n d f 2 is s p l i t o ff ( H e r s h e y & T h a c h , 19 67; K o la f o f s k y , D e w e y ,
H e r s h e y & T h a c h , 1968).
I;
7
F a c to r fg i s a l s o c l a s s i f i e d a s a n i n i t i a t i o n f a c to r ( R e v e l, L e l o n g ,
B raw erm an & G r o s , 1968), h o w e v e r i t s e x a c t fu n c tio n re m a in s in q u e s ­
tio n .
S e v e ra l g ro u p s (R a v e l, S h o r e y , G a r n e r , D a w k in s & S h i v e , 19 69;
K a e m p fe r, 1971) h a v e i m p lic a te d fg a s a s p e c i f i c r e c o g n itio n p ro te in in
n a t u r a l mRNA i n i t i a t i o n .
D u b n o ff & M a itra (19 69) h a v e re p o rte d th a t in
a d d itio n to s tim u la tin g a n a t u r a l mRNA s y s t e m , fg c a u s e s th e d i s s o c i ­
a tio n o f fre e 70s r ib o s o m e s in to 50s a n d 8 0 s s u b u n i t s . In a d d itio n ,
th e y d e m o n s t r a t e d t h a t fg i s a l s o re q u ire d for c o m p le x (I) f o r m a tio n .
P e rh a p s th e m o s t c o n v in c in g m o d el o f a c t i o n of fg c o m e s from Kaempfer
(1971) w ho h a s sh o w n t h a t f a c to r fg p r e v e n t s th e fo rm a tio n o f 70s r i b o s o m a l p a r t i c l e s from 5 0 s a n d 8 0 s s u b u n i t s . K a e m p fe r's w ork a l s o h a s
sh o w n an i n v e r s e r e l a t i o n s h i p b e tw e e n th e r a te o f p r o te in s y n t h e s i s a n d
th e c o n c e n tr a tio n o f fre e 70s r i b o s o m e s .
He c o n c lu d e s t h a t fg s e r v e s
to r e g u la te th e r a te o f i n i t i a t i o n o f p r o te in s y n t h e s i s . W h en th e r a te o f
p ro te in s y n t h e s i s i s h i g h , th e c o n c e n tr a tio n o f fg d e c r e a s e s , due to i t s
u s e in c o m p le x (I) fo r m a tio n . C o n s e q u e n t l y , th e 50s a n d 8 0 s s u b u n its
r e l e a s e d d u rin g p e p t i d e te r m in a tio n form 70s r ib o s o m e s w h ic h a re i n ­
a c t i v e in p e p tid e i n i t i a t i o n .
The r a te o f p ro te in s y n t h e s i s m ay th e n
d ro p , t h e c o n c e n t r a t i o n o f free fg i n c r e a s e s , d i s s o c i a t i n g th e 70s to
fre e 50s a n d 8 0 s s u b u n i t s w h ic h c an a g a i n i n i t i a t e p r o te in s y n t h e s i s .
In a n y c a s e , w ith th e fo rm a tio n o f c o m p le x (III), th e in i t i a t i o n
p r o c e s s e n d s . In t h i s s t a t e th e "P" s i t e is f ille d w ith f-m et-tR N A , a n d
th e a d j a c e n t "A" s i t e i s e m p ty . The p r o c e s s o f e l o n g a t io n now e n s u e s .
8
M a n y a n tib io tic s e x e r t t h e i r e f f e c t by in h ib iti n g th e i n i t i a t i o n p ro ­
cess.
The t e t r a c y c l i n e s a n d l i n c o s i n a m i d e s a b o l i s h b a c t e r i a l p ro te in
s y n t h e s i s by in h ib iti n g th e b in d in g o f aa-tR N A -to r i b o s o m e s . The t e t r a ­
c y c l i n e s b in d s p e c i f i c a l l y to th e 30s rib o s o m a l s u b u n it a n d p re s u m a b ly
b lo c k c o m p le x (I) fo rm a tio n (S u arez & N a t h a n s , 19 65). L in co m y c in
b in d s s p e c i f i c a l l y to th e 5 0 s s u b u n i t , a n d p e r h a p s b lo c k s c o m p le x (II)
fo rm atio n (C h a n g , Sih & W e i s b l u m , 1 966).
A lthough n o t c l a s s e d as. a d r u g , G M P -PC P ( S '- g u a n y lm e th e le n e d ip h o s p h o n a te ) c a n b lo c k fo rm atio n o f c o m p le x (III) (T h ach & T h a c h ,
1 9 7 1 ). This co m p lex ' r e q u ir e s th e h y d r o ly s is o f GTP to GDP a n d i n o r ­
g a n ic p h o s p h a te for i t s fo rm a tio n . G M P -P C P w ill b in d in p l a c e o f G TP,
b u t s i n c e i t c a n n o t b e h y d r o l y z e d , th e c o m p le x i s c o m p e t itiv e ly i n ­
h ib ite d .
E lo n g a tio n
/
•
-
I m m e d ia te ly a f t e r th e fo rm atio n o f c o m p le x (III), e lo n g a t io n p ro ­
c e e d s w ith th e b in d in g o f t r a n s f e r f a c t o r T u , GTP, a n d th e s e c o n d 1
.,aa-tRNA (in th e i l l u s t r a t i o n i t is val-tRN A ) to form c o m p le x (IV) (T h ach
& T h a c h , 1 9 7 1 ). In t h i s s t a t e , th e aa-tR N A i s b o und to t h e " p r e A" s i t e
( a n a la g o u s to the. " p re P" s i t e ) . T u , in a m a n n er a n a lo g o u s to f g , c a t a ­
l y z e s th e h y d r o ly s is o f GTP th u s " a c c o m m o d a tin g " th e n e w aa-tR N A a n d
form ing c o m p le x (V).
W ith th e tw o aa-tR N A m o le c u le s in c o r r e c t s t e r i c a p p o s i t i o n to
th e p e p tid y l t r a n s f e r a s e e n z y m e , p e p tid e b o n d fo rm atio n o c c u r s . The
9
r e s u l t o f t h i s , c o m p le x (VI), r e t a i n s th e f - m e t- v a l- tR N A in th e "A" s i t e ,
a n d a fre e tRNA in th e "P" s i t e .
C o m p le x (VI) i s now in a p o s i t i o n to u n d erg o t r a n s l o c a t i o n . Al­
th o u g h s e v e r a l i n t e r m e d i a t e s m ay be i n v o l v e d , th e h y d r o ly s is o f GTP
c a t a l y z e d by e lo n g a t io n f a c to r G c a u s e s th e m o v e m e n t o f th e mRNA
t h r e e n u c l e o t i d e s in th e 5' d i r e c t i o n ( W a ts o n , 19 64). The n e x t t r i p l e t in
s e q u e n c e i s th e n m ade a v a i l a b l e for r e c o g n itio n b y th e " p r e A" s i t e .
C o m p le x (VII) i s form ed w ith th e s i m u lta n e o u s r e l e a s e o f free
tRNA from t h e "P" s i t e , a n d th e "P" s i t e now c o n ta i n s f - m e t- v a l- tR N A . •
The c o m p le x i s n o w in a s t a t e a n a l a g o u s . t o t h a t o f c o m p le x (III), a n d a
n e w s p e c i f i c aa-tR N A m ay b in d to th e n o w v a c a n t " p r e A" s i t e . E lo n g a ­
tio n n o w p r o c e e d s in a c y c l i c f a s h io n for e a c h s u c c e s s i v e c o d o n u n til a
te r m in a tio n c o d o n i s e n c o u n t e r e d .
■ A w h o le h o s t o f a g e n t s h a v e b e e n sh o w n to b lo c k p e p tid e e l o n g a ­
t i o n . G M P -P C P w ill a l s o b lo c k fo rm atio n o f c o m p le x e s (V) a n d (VII), a s
b o th r e q u ire h y d r o ly s is o f GTP for t h e i r fo rm a tio n . F u s c i d i c a c i d a l s o
i n h i b i t s th e t r a n s i t i o n from c o m p le x (VI) to (VII) by in h i b i t i n g G fa c to r
(T a n a k a , K ih o s h ita & M a s u k a w a , 19 6 8 ). P u rom ycin c a n r e a c t w ith a n y
c o m p le x ( I II)- Iik e m o n o s o m e , form ing a n a a - p u r o m y c in m o le c u le w h ic h
d i s s o c i a t e s from th e c o m p le x p re m a tu re ly te r m in a tin g p r o te in s y n t h e s i s
( M o r r i s , 1961). The a m in o g ly c o s id e a n t i b i o t i c s , s t r e p t o m y c i n , k a n a m y c in , a n d n e o m y c in p r o d u c e , am ong o th e r t h i n g s , s p e c i f i c m i s r e a d ­
in g s in th e g e n e t i c c o d e d u rin g e lo n g a t io n ( D a v i e s , G ilb e r t & G o r i n i ,
10
19 6 4 ). A fter a t t a c h i n g to th e r i b o s o m e , t h e s e d ru g s a p p e a r to p erm it
i n c o r p o r a tio n o f o n e o r m ore i n c o r r e c t a m in o a c id s i n to a g ro w in g p e p ­
t i d e c h a i n , r e s u l t i n g in s y n t h e s i s o f d e f e c t i v e p r o t e i n s . z
T e rm in a tio n *I
It w a s found b y th e s tu d y o f s p e c i f i c mRNA m u ta n ts t h a t c e r ta in
c o d o n s c a u s e d te r m in a tio n o f p e p t i d e s y n t h e s i s . W h en c o d o n s UAA
(o c h re ), UAG (am ber), a n d UGA (umber) w e re re a d by th e rib o s o m a l c o m ­
p l e x , p r o te in s y n t h e s i s w a s te r m in a te d b y r e l e a s e o f th e p e p tid e from
th e r ib o s o m a l b o u n d tRNA (G a r e n , 1968; L a s t , S t a n l e y , S a l a s , e t a l . ,
I
'
.
1 9 6 7 ).
F a c to r s n e c e s s a r y for te r m in a tio n in n a tu r a l m e s s e n g e r RNA s y s ­
te m s h a v e b e e n r e p o r te d . F a c to r R^ i s s p e c i f i c for te r m in a tio n c o d o n s
UAA or UAG, a n d F a c to r R2 for c o d o n s UAA or UGA ( C a p e c c h i , 19 67;
C a s k e y , T o m k in s , S c o l n i c k , C a r f k & N i r e n b e r g , 1968; Tom kins &
N i r e n b e r g , 1968). The a b s e n c e of t h e s e r e l e a s e f a c to r s c a u s e s i n h i b i ­
tio n o f p r o te in s y n t h e s i s by p r e v e n tin g r e l e a s e of th e c o m p le te d p e p tid e
c h a i n . Upon r e l e a s e o f th e g ro w in g p e p t i d e c h a i n , th e r ib o s o m a l c o m r
p l e x d i s s o c i a t e s , p ro d u c in g fre e 3 0 s a n d 5 0 s rib o s o m a l s u b u n i t s , a s
w e ll a s fre e mRNA. The 30s r ib o s o m a l s u b u n i t m ay n o w a g a i n i n i t i a t e
p ro te in s y n t h e s i s , or form t r a n s i e n t 70s u n its (K a em p fer, 1971).
/
I
11
(d) C h lo ra m p h e n ic o l in p ro te in s y n t h e s i s
C h lo r a m p h e n ic o l1s mode o f a c t i o n h a s e lu d e d s c o rp s o f i n v e s t i ­
g a to r s s i n c e i t s d i s c o v e r y in a s o i l s a m p le o b ta in e d from V e n e z u e la in
1 9 4 3 . O r ig in a lly e x c r e t e d b y S tr e p to m y c e s v e n e z u e l a e , c h lo r a m p h e n i­
c o l e a r n e d th e d i s t i n c t i o n o f b e in g th e f i r s t c o m m e rc ia lly s y n t h e s i z e d
drug (C o n t r o u l i s , 1949). Its a b i l i t y to i n h i b i t p ro te in s y n t h e s i s in b a c ­
t e r i a ( W i s s e m a n 7 H a n n 7 H o o p s & S m a d e l7 19 62) le d to i t s common u s e
in m o le c u la r b io lo g y . For t h i s r e a s o n i t i s h ig h ly d e s i r a b l e to kn o w its
p r e c i s e m o le c u la r m e c h a n is m o f a c t i o n .
C h lo ra m p h e n ic o l is a n in c r e d i b l y e f f e c t i v e drug a g a i n s t Gram
p o s i t i v e a n d n e g a ti v e b a c t e r i a , b l u e - g r e e n a lg a e a n d c e r t a i n v i r u s e s
( K o z in s k y 7 19 63), w h ile re m a in in g q u ite in e f f e c t i v e a g a i n s t m am m alian
c e l l s ( V a z q u e z , 19 66). It i s a r e l a t e d m em ber o f th e e p h e d r a s e r i e s of
a l k a l o i d s , w ith a n a c c e p t e d c h e m ic a l n am e o f D ( - ) t h r e o - 2 d ic h lo ro a c e ta m id o -l-p -n itro p h e n y l-1 ,3 -p ro p a n e d io l, and w as first com ­
m e r c ia l ly s y n t h e s i z e d b y Long & Troutm an in 1 949. Its s tr u c tu r e is
sh o w n in F ig . 2.
The f ir s t m ode o f a c t i o n e x p e r im e n t im p lic a te d i t s in h ib ito r y a c •tion on b a c t e r i a l e s t e r a s e s (S m ith , W o rre l & S w a n s o n 7 1949), b u t i t w a s
l e f t to G a le a n d P a in e in 19 51 to g iv e th e f i r s t c lu e t h a t c h lo r a m p h e n i­
c o l i n h i b i t e d p ro te in m e ta b o lis m . In a n o w c l a s s i c e x p e r im e n t , H ahn
a n d W is s e m a n in 1951 s h o w e d t h a t c h lo ra m p h e n ic o l i n h i b i t e d th e
.12
fo rm a tio n o f a d a p ti v e e n z y m e s . S in c e t h a t tim e , i t h a s b e e n g e n e r a lly
a c c e p t e d t h a t c h lo r a m p h e n ic o l i n h i b i t s p r o te in s y n t h e s i s .
S tr u c tu r a l s t u d i e s on th e d rug s h o w t h a t th e d i c h l o r o a c e t a m i d o ,
h y d ro x y ls a n d p ro p a n o l g ro u p s a re n e c e s s a r y for b io lo g ic a c t i v i t y
(B ro c k , 1961; Sm ith & H in m a n , 1953; W o o l e y , 1950). The n itro group
n e e d o n ly be a p o la r iz in g group for a c t i v i t y (B ro c k , 1 9 6 1 ). The a m id e
b o n d a n d h y d ro x y ls a re th o u g h t to be th e e s s e n t i a l c o n s t i t u e n t s for b i o ­
l o g i c a l a c t i v i t y , s i n c e a l t e r a t i o n o f t h e s e m o ie tie s r e d u c e s th e b i o l o g i ­
c a l a c t i v i t y to z e r o (B ro c k , 1 961). I t i s o f fu rth e r i n t e r e s t t h a t , o f th e
fo u r i s o m e r s p o s s i b l e , o n ly t h e D ( - ) th r e o h a s b io l o g i c a l a c t i v i t y (H ahn
& W i s s e m a n , 1 951).
S in c e 19 5 1 , e x p e r im e n t s h a v e c e n t e r e d on f in d i n g th e e x a c t
m o le c u la r m e c h a n is m o f a c t i o n . It i s n o w know n t h a t c h lo r a m p h e n ic o l
d o e s n o t i n h i b i t am in o a c i d a c t i v a t i o n (D e M o s s & N o v e l l ! , 1955), d o e s
n o t i n h i b i t fo rm atio n o f th e am in o a c y l tRNA c o m p le x (L a c k s & G ros ,
1959), a n d d o e s n o t h a v e a n y e f f e c t on DNA or RNA s y n t h e s i s ( G a le ,
1 9 6 2 ) . F u r th e r m o r e , c h lo r a m p h e n ic o l d o e s n o t i n h i b i t t h e b in d in g o f
tRNA to r ib o s o m e s ( C a n n o n , Krug & G i l b e r t , 19 63; J a r d e tz k y & J u l i a n ,
1964) a n d i t d o e s n o t p r e v e n t th e r e l e a s e o f c o m p le te d p r o te in ( D a s ,
G o l d s t e i n & K a n n e r , 1 966).
B inding s t u d i e s h a v e sh o w n t h a t c h lo ra m p h e n ic o l b in d s w e a k ly
b u t s p e c i f i c a l l y to t h e 5 0 s rib o s o m a l s u b u n i t ( V a z q u e z , 19 6 6 a , 19 64),
a n d s u c h b in d in g o c c u r s im m e d ia te ly upon th e in c u b a t io n o f rib o s o m e s
J
'
13
a n d c h l o r a m p h e n i c o l . The b in d in g r e q u ir e s no e n e r g y a n d i s r e v e r s i b le
(L e h n i n g e r , 19 61). S u c h s t u d i e s s h o w a c h lo ra m p h e n ic o l- rib o s 'o m e
b in d in g r a tio o f 1:1 a t c h lo r a m p h e n ic o l c o n c e n t r a t i o n s b e lo w 50 |_ig./ml.
(W o lfe & H ahn, 1965), a n d a b in d in g r a tio o f 2:1 a t c o n c e n t r a t i o n s e x ­
c e e d i n g 2 00
|J .g . / m
l . ( D a s , G o ld s te i n & K a n n e r , 19 65). The im p lic a tio n
o f tw o d if f e r e n t b in d in g c o n f ig u r a tio n s i s n o t c l e a r .
I t w o u ld a p p e a r o b v io u s from th e l i t e r a t u r e t h a t c h lo ra m p h e n ic o l
i n h ib iti o n i s d u e to i n t e r f e r e n c e w ith p r o te in s y n t h e s i s . H o w e v e r , e x ­
tre m e c a r e m u s t b e t a k e n in th e i n t e r p r e t a t i o n o f s u c h d a t a .
C h lo ra m ­
p h e n ic o l h a s b e e n s h o w n to b e a p o t e n t i n h ib ito r o f g l u c o s e transfer in
t e i c h o i c a c i d b i o s y n t h e s i s , p o w e rfu lly in h ib iti n g c e l l w a ll s y n t h e s i s in
c o n c e n t r a t i o n s o f c h lo r a m p h e n ic o l o f 500 p g . / m l . (S to w , 1 9 7 1 ). Due to
t h e d i f f e r e n t i a l p e r m e a b i l i t y of c h lo r a m p h e n ic o l am ong m ic ro o rg a n is m s ,
i t i s d if f i c u l t to e x t r a p o l a t e a n d c o m p a re t h e m any in v iv o e f f e c t s ,of
ch lo ra m p h en ico l.
The in v iv o c o n c e n t r a t i o n s o f c h lo ra m p h e n ic o l r e q u ir e d for 50%
in h i b i t i o n ra n g e from 0 .3 8 p ig ./m l. for H e m o p h ilu s i n f l u e n z a e to o v e r
100 p ig ./m l. for P s e u d o m o n a s . E s c h e r i c h i a c o ll a lo n e v a r i e s b e tw e e n 3.1
a n d 100+ iag./ m l ., w ith a m e d ia n o f 6.2 |a g ./m l. (K agan, 19 7 0 ). I n d e e d ,
s u c h a w id e ra n g e o f e f f e c t i v e c h lo ra m p h e n ic o l c o n c e n t r a t i o n s in E_. c o li
m ig h t r e f l e c t s u b t l e d i f f e r e n c e s in m e c h a n is m s of i n h ib iti o n o f p ro te in
s y n t h e s i s , a s w e ll a s d i f f e r e n c e s , i n drug p e r m e a b il ity .
14
In v itr o s t u d i e s u s in g p u rif ie d r ib o s o m e s a g a in s h o w a w id e ra n g e
in t h e e f f e c t i v e n e s s o f c h l o r a m p h e n i c o l . U s in g 60% i n h ib iti b n o f to t a l
14
C a c i d p r e c i p i t a b l e c o u n ts a s a r e f e r e n c e , a p o ly A s y s t e m r e q u ire s
67 |-ig ./m l. o f c h lo ra m p h e n ic o l ( J u l i a n , 19 65), w h e r e a s a n MS2 mRNAd i r e c t e d s y s te m r e q u ir e s only^Z p g . / m l . o f c h lo ra m p h e n ic o l ( s e e "Ex­
p e r im e n ta l R e s u l t s " ) .
i.
/
2.
EXPERIMENTAL RATIONALE
MS2 v ir u s i s a n ic o s a h e d r 'a l s h a p e d b a c t e r i o p h a g e o f 25 m i l l i ­
m ic ro n s d i a m e t e r , a m o le c u la r w e ig h t o f a b o u t 3.5 m illio n d a lto n s , a n d
a s e d i m e n t a t i o n c o n s t a n t o f 81 S v e d b u rg u n its (S tr a u s s & S i n s h e i m e r ,
19 6 3 ). It h a s a n in fo rm a tio n c o n t e n t in i t s s i n g l e s t r a n d e d RNA core
s u f f i c i e n t to c o d e fo r th r e e p r o te in s ; th e c o a t p r o t e i n , a m a tu ra tio n
f a c t o r , a n d a n RNA d e p e n d e n t RNA p o ly m e r a s e (C a p e c c h i , 19 66).
W h en p u rif ie d MS2 RNA i s p l a c e d in to a n in v itr o p r o te in s y n t h e ­
s i z i n g s y s t e m , o v e r t e n tim e s a s m u ch c o a t p ro te in i s p r o d u c e d a s th e
o t h e r tw o p r o te in s ( W a r d , 1968; K o la k o f s k y , D e w e y , H e r s h e y & T h a c h ,
19 6 8 ). If th e a m in o a c i d h i s t i d i n e i s l e f t o u t o f th e in c u b a t i o n m ix tu r e ,
o n ly c o a t p r o te in s h o u ld b e p ro d u c e d ( N a t h a n s , N o t a n i , S c h w a rz &
Z i n d e r , 1 9 6 2 ).
S u c h a s y s te m a llo w s t h e in v itr o p ro d u c tio n o f a u n iq u e p ro te in
w ith a k n o w n a m in o a c i d s e q u e n c e (W e b er & K o n ig s b e r g , 19 67; L in ,
T su n g & F r a e n k e l - C o n r a t , 19 67). F u r th e r m o r e , i t h a s b e e n sh o w n t h a t
th e in v itr o p r o d u c t i s e s s e n t i a l l y th e s a m e a s th e n a t i v e c o a t p ro te in
m o le c u le ( N a t h a n s , N o t a n i , S c h w a rz & Z i n d e r , 19 62; N a t h a n s , O e s c h g e r ,
E g g en & S h im u r a , 19 6 6 ). The a m in o a c i d s e q u e n c e o f MS2 c o a t p ro te in
i s s h o w n in F ig . 3. A c o n v e n i e n t y e t h ig h ly r e p r o d u c ib le a n d e x a c t
m e th o d for a s s a y i n g th e MS2 in v itr o c o a t p ro te in m ay b e g l e a n e d from
t h e t e c h n i q u e s d e v e lo p e d b y K a tz , D re y e r & A n fin se n in 19 59.
16
F in g e rp r in tin g i n v o lv e s th e re p r o d u c ib le o x id a tio n a n d e n z y m a tic
d i g e s t i o n o f a p r o te in to s m a l l e r p e p t i d e s .
The p ro te in is f i r s t o x id iz e d
w ith p e ro x y fo rm ic a c i d , w h ic h ru p tu r e s d i s u l f i d e b o n d s a n d u s u a ll y i n ­
c r e a s e s t h e s o l u b i l i t y in a q u e o u s s o l v e n t s .
H y d r o ly s is w ith try p s in is
n e x t e m p lo y e d , w h ic h c l e a v e s th e p r o te in a t t h e c a r b o x y l e n d s o f l y s i n e
a n d a r g i n i n e . This p r o c e d u r e p r o d u c e s a d i s t i n c t s e t o f s m a l le r p e p ­
t i d e s w ith a d i s t r i b u t i o n , s i z e , a n d p r o p e r tie s c h a r a c t e r i s t i c o f th e
o r ig i n a l c o a t p r o t e i n .
T h e s e t r y p t i c p e p t i d e s , e l e v e n in th e c a s e o f MS2 c o a t p r o te in ,
a r e d i s p l a y e d a s a tw o - d i m e n s i o n a l m a p , th e f in g e r p r in t. The p e p t i d e s
r e s u l t i n g from th e h y d r o l y s i s o f th e c o a t p ro te in a re f i r s t s e p a r a t e d by
h i g h - v o l t a g e e l e c t r o p h o r e s i s on a s h e e t o f f i l t e r p a p e r , a n d s e c o n d ly
b y a s c e n d i n g c h ro m a to g ra p h y in a d i r e c t i o n 9 0 ° to t h a t u s e d during
e l e c t r o p h o r e s i s . The r e s u l t is a t w o - d i m e n s i o n a l d i s p l a y o f e le v e n
(
s p o t s w h ic h h a v e b e e n s e p a r a t e d b y s i z e a n d c h a r g e , a p d m ade v i s i b l e
b y s p r a y in g w ith n in h y d r i n , a r e a g e n t w h ic h r e a c t s w ith a m in o g ro u p s
p r o d u c in g a v i s i b l e c o lo r .
A fin g e r p r in t c a n a l s o b e m ad e o f th e in v itro MS2 c o a t p ro te in by
■*'
. 1 4
i n i t i a l l y la b e l i n g th e in v itr o p ro te in w ith
i s th e n f in g e r p r in te d . To v i s u a l i z e t h e
C -a m in o a c i d s . The p ro te in
14
. C - t r y p t i c p e p t i d e s , a s h e e t of
u n e x p o s e d X -ra y film i s e x p o s e d to th e fin g e rp r in t for a b o u t a w e e k ,
a n d th e X -ra y film d e v e l o p e d . E ach r a d i o a c t i v e tr y p t i c p e p tid e w ill p ro ­
d u c e a r a d i o - o p a q u e s p o t on th e X -ra y film . In t h i s c a s e , o n ly te n
17
s p o t s s h o u ld o c c u r a s p e p tid e n u m b e r e l e v e n w ill n o t h a v e a n y ^ C a m in o a c i d a s s o c i a t e d w ith i t , i f
14
' 14
C - I y s i n e a n d C - a r g i n i n e a re u s e d
a s th e o n ly r a d i o a c t i v e l a b e l s .
The t r a n s l a t i o n a t a n y p o in t in tim e o f th e c o a t p r o te in c i s t r o n of
th e MS2 v iru s n a tu r a l m RNA may be e n v i s i o n e d a s in v o lv in g a p o p u l a ­
tio n o f r i b o s o m e / m e s s e n g e r c o m p le x e s in a l l s t a g e s o f t r a n s l a t i o n .
C o n s e q u e n t l y , e v e r y c o m p le x 'w ill h a v e in c o r p o r a te d an N - te r m i n a l
a m in o a c i d , b u t o n ly th o s e c o m p le x e s in th e fin a l s t a g e s o f p o ly p e p tid e
s y n t h e s i s w ill h a v e in c o r p o r a te d a C - te r m in a l am ino a c i d . I f c h lo ra m ­
p h e n ic o l (CAP) w e re to b lo c k a s t e p in e l o n g a t i o n , w e w o u ld e x p e c t a
p o p u la tio n o f r i b o s o m e / m e s s e n g e r c o m p le x e s fro z e n in th e v a rio u s
s t a g e s o f e lo n g a t i o n . H e n c e , w e w o u ld n o t e x p e c t a n y d e c r e a s e in th e
rib o s o m a l
14
■
C n a s c e n t p e p t i d e s a s c o m p a re d to a n o rm a lly o c c u r rin g .
p r o te in s y n t h e s i z i n g s y s t e m . F u r th e r m o re , a s e lo n g a t io n w a s in h ib ite d
on a
mRNA s t r a n d , a n i n c r e a s e in th e q u a n tity o f th e N - te r m i n a l^ p e p ­
t i d e s m ig h t be e x p e c te d a s n e w ly i n i t i a t e d rib o s o m e s w e re a llo w e d to
s y n t h e s i z e p e p t i d e s b e fo re b e in g i n h i b i t e d in som e s t a g e o f e lo n g a t io n .
N a th a n s (1965) e x a m in e d th e e f f e c t o f CAP on th e r e l e a s e d ^Re­
l a b e l e d MS2 in v itr o p e p t i d e s . H is m e th o d o lo g y u t i l i z e d a fin g e rp rin t
o f a tr y p t i c d i g e s t o f th e n o n - r ib o s o m a l b o u n d
p e p tid e s.
14
C - I a b e l e d MS2 in v itro
He found no i n c r e a s e in th e tr y p t ic N - t e r m i n a l p e p t i d e s o v e r
th e C - te r m in a l p e p t i d e s w h e n s u c h p e p t i d e s w e re s y n t h e s i z e d in th e
p r e s e n c e o f 3.5 [ig ./m l. o f CAP. T h is w a s in c o n t r a s t to th e r e l e a s e d
18
p e p t i d e s form ed in th e p r e s e n c e o f p u ro m y c in , w h ic h s h o w e d a n i n ­
c r e a s e in th e n u m b e r o f N - t e r m i n a l p e p t i d e s .
T h e s e r e s u l t s , w e re in
a c c o r d w ith a n e lo n g a t io n i n h ib iti o n m o d e l for CAP, a s o n e w o u ld e x ­
p e c t t h e r e l e a s e d p e p t i d e s to b e w h o le a n d p o s s e s s an e q u a l n u m b er o f
N - and C -term in al p e p tid e s .
H is w ork a l s o te n d e d to ru le o u t CAP i n ­
d u c e d p re m a tu re r e l e a s e , for in t h i s c a s e w e s h o u ld e x p e c t r e s u l t s
s i m i l a r t o t h o s e s e e n w ith th e r e l e a s e d p e p t i d e s s y n t h e s i z e d in th e
p r e s e n c e o f p u ro m y c in .
H o w e v e r, h e d id n o t e x a m in e CAP'S e f f e c t on
t h e r ib o s o m a l b o u n d ^ C - n a s c e n t p e p t i d e s . I t i s h e re t h a t th e tru e t e s t
fo r a n e lo n g a t io n i n h ib iti o n m o d el e x i s t s , s i n c e . t h e p e p t i d e s r e s u l t i n g
from i n h ib iti o n w o u ld l i k e l y b e tr a p p e d on th e m e s s e n g e r - r i b o s o m e c o m ­
p l e x . I t w a s th u s d e c i d e d to co m p are t h e r ib o s o m a l b o u n d to r e l e a s e d
p r o d u c ts u s in g N a t h a n 's p r o c e d u r e s .
I
I
3.
MATERIALS AND METHODS
(a)
B a c te r ia l a n d b a c te r io p h a g e s t r a i n s
E s c h e r i c h i a c o ll Q-13 w a s o b ta in e d from G e n e r a l B io c h e m ic a ls in
l a t e lo g p h a s e , u n w a s h e d . The b a c t e r i a w e re d e l i v e r e d fro z e n in 4 0 - g .
c h u n k s a n d s t o r e d a t -IO0 C u n til u s e d .
C a t . #150070.
E . c o li Q-13 w a s
a l s o o b ta in e d from D r. G . S t r e i s i n g e r o f th e U n iv e r s ity o f O re g o n ,
v
E u g e n e . S u ch b a c t e r i a w e re u s e d a s a s o u r c e o f r i b o s o m e s , S -3 0 e x ­
t r a c t s a n d a s a v ir u s h o s t .
E s c h e r ic h ia c o li A-19 w a s o b ta in e d from
D r. J. C la rk o f th e U n i v e r s i t y o f I l l i n o i s , U r b a n a , a n d u s e d for S -3 0
e x t r a c t s a n d a s a v ir u s h o s t .
h o st.
E s c h e r ic h ia c o li K-12 w a s u s e d a s a v ir u s
E s c h e r i c h i a c o li B w a s u s e d a s a h o s t d i f f e r e n tia tin g p h a g e T^
a n d M S 2 . E s c h e r i c h i a c o l i M RE-600 w a s o b ta in e d from D r. J . D a v ie s o f
th e U n i v e r s i t y o f W i s c o n s i n , M a d i s o n . It w a s grow n on m e d ia LS and
u s e d for th e p r e p a r a t io n o f i n i t i a t i o n f a c to r s a n d w a s h e d r i b o s o m e s .
B a c te rio p h a g e M S2. w a s o b ta in e d a s a ly o p h y liz e d p o w d e r from
t h e A m erican Type C u ltu re A s s o c i a t i o n , R o c k v ille , M a r y la n d .
C at.
# 1 5 5 9 7 -B . It w a s u s e d fo r th e p r e p a r a t io n o f v iru s mRNA a n d c o a t p r o ­
te in .
I
(b)
B a c te ria l a n d v ir a l g ro w th m e d ia
All w e ig h ts r e f e r to g ram s p e r l i t e r o f s in g le d i s t i l l e d w a te r . All
m e d ia w e re a u t o c l a v e d for 30 m in u te s a t 15 to 20# o f s t e a m .
20
TYS+ M edium
10 g . B a c to - tr y p to n e
1 g . B a c to -y e a st e x tra ct
S g . so d iu m c h lo rid e
A fter a u t o c l a v i n g , th e fo llo w in g ( e a c h a u t o c l a v e d s e p a r a te ly )
w e re a d d e d u s in g s t e r i l e te c h n iq u e :
10 m l. o f 10% g l u c o s e
2 m l. o f I M c a lc iu m c h lo rid e
I m l. o f th ia m in e h y d ro c h lo rid e (10 m g . / m l . )
(TYS m edium l e s s th e g l u c o s e , c a lc iu m a n d th ia m in e w a s d e s i g ­
n a t e d TYS . U pon th e a d d itio n of t h e s e s o l u t i o n s , TYS .)
Top L a y e r
TYS+ m edium w ith 4 gm . / l i t e r B a c to - a g a r
Agar P l a t e s a n d S la n ts
TYS
m edium w ith 15 g . / l i t e r B a c t o - a g a r
S e r ia l D ilu tio n S o lu tio n
TYS
m edium
LS M edium
4.0 g . n u tr i e n t b ro th
4.0 g . N a C l
0.5 g . g lu c o s e
0.5 g . y e a s t e x t r a c t
I
V
21
1.0 g . KH2 PO4
0.5 g . C a C l 2
0.5 g . M g C l2
(c) Buffers
All b u ff e rs w e re m ad e w ith d o u b l y - d i s t i l l e d w a t e r , th e s e c o n d
d i s t i l l a t i o n o f ta p d i s t i l l e d w a t e r o c c u r rin g in a C o rn in g m o d el AG-2
d i s t i l l a t i o n a p p a r a t u s . P e rio d ic s a m p l e s o f t h i s w a te r r e v e a l e d a m in imum s p e c i f i c r e s i s t a n c e o f 800 K n c m
.
All p H a d j u s t m e n t s w e re m ad e w ith d i s t i l l e d h y d r o c h lo r ic a c i d .
S u c h a c i d w a s th e m id d le th ird d i s t i l l a t e o f a 1:2 d ilu tio n o f d i s t i l l e d
w a t e r w ith r e a g e n t h y d r o c h lo r ic a c i d .
M e a s u r e m e n ts o f p H w e re m ade
on a C o rn in g m o d e l 12 pH m e te r , a f t e r 'p r i o r s t a n d a r d i z a t i o n u s in g
/
p H y d rio n t a b l e t s .
B e n t o n ite , a c o l l o i d a l a lu m in u m s i l i c a t e c l a y , w a s p re p a r e d by
t h e m e th o d o f D unn & H itc h b o m (19 6 5 ). Such w a s h e d b e n to n ite s u s p e n
s i o n s h a v e b e e n n o te d for t h e i r a d s o r b a n t p r o p e r tie s in re m o v in g r i b o n u c l e a s e s from a q u e o u s s o l u t i o n s . Two d ro p s o f b e n t o n i t e s u s p e n s i o n
p e r l i t e r o f b u ffe r w a s a d d e d a n d th e r e s u l t i n g d i s p e r s i o n a llo w e d to
s t a n d a t 20 C fo r a t l e a s t tw o h o u r s . The b u ffe r w a s t h e n f il te r e d
th ro u g h M illip o r e HAWP00010 5 0 -m m . n i t r o c e l l u l o s e f i l t e r s in to a
s t e r i l e r i b o n u c l e a s e - f r e e r e a g e n t b o t t l e u s in g a n a u t o c l a v e d f i l t r a ­
tio n a p p a r a t u s .
I
22
All b u ff e rs w e re s to r e d a t 2 ° C u n l e s s o th e r w is e i n d i c a t e d .
Buffer A^fg
0.02 M Tris
0.01 M M g(O A c)2
• 0.01 M p - m e r c a p to e th a n o l (a d d e d j u s t b e fo re u s e )
The b u ffe r w a s a d j u s t e d to pH 7.4 a t room te m p e ra tu re w ith d is
t i l l e d h y d r o c h lo r ic a c i d , th e n b e n to n ite t r e a t e d a n d f i l t e r e d .
Buffer A Fr,
________ s_z_
0.05 M Tris '
0.001 M d i t h i o t h r e i t o l (a d d e d j u s t p rio r to u se)
N H y1SOz1 (e n z y m e g r a d e , a d d e d to s a t u r a t i o n a t 2°C )
The b u ffe r w a s a d j u s t e d to p H 7.4 a t 2 ° C w ith d i s t i l l e d h y d ro ­
c h l o r i c a c i d th e n b e n to n ite t r e a t e d a n d f il te r e d . NH^SO^ w a s th e n
a d d e d to s a t u r a t i o n a t 2 ° C .
Buffer B^fg
1.0 M N H 4 Cl
0.02 M Tris
0.002 M Mg(OAc)2
0.002 M d it h i o t h r e i t o l (a d d e d j u s t p rio r to u se)
The b u ffe r w a s a d ju s te d to pH 7.8 a t 2 °C w ith d i s t i l l e d h y d ro ­
c h lo r ic a c id th e n b e n to n ite tr e a te d a n d f ilte r e d .
23
Buffer CBN-I
P y rid in e : 50 p a rts
A c e tic a c id : 15 p a rts
n -b u ta n o l; 75 p a rts
g la s s d i s tille d w a te r: 60 p a rts
(All p a rts by volum e)
S to red a t room te m p e r a tu r e .
B uffer EBN-I
P y rid in e : 1.2%
,
9
. A c e tic a c id ; 1.3%
n -b u ta n o l: 2.5%
>
/
G la s s d i s tille d w a te r: 95%
(All p e r c e n ta g e s b y volum e)
S to red a t room te m p e r a tu r e .
B uffer H i-IFE M
0.002 M M g C l2
1.0 M N H 4 C l
0.01 M Tris
0.001 M d ith io th r e ito l (a d d e d b e fo re filte rin g )
The b u ffe r w a s a d j u s t e d to pH 7.5 w ith d i s t i l l e d h y d ro c h lo ric a c id
t h e n b e n to n ite t r e a t e d a n d f i l t e r e d .
24
B uffer IFE M -I
0,002 M M g C l2
0.02 5 M N H 4 C l
0.01 M T ris
0.001 M d ith io th r e ito l (a d d e d j u s t p rio r to u se )
The b u ffe r w a s a d ju s te d to pH 7.5 a t 2 °C w ith d i s t i l l e d h y d ro ­
c h lo r ic a c id th e n b e n to n ite tr e a te d a n d f il te r e d .
B uffer IFEM -2
0.002 M M g C l2
0.100 M N H 4 C l
0.010 M T ris
0.001 M d ith io th r e ito l (a d d e d j u s t p rio r to u se )
(
The b u ffe r w a s a d ju s te d to pH 7.5 a t 2 °C w ith d i s t i l l e d h y d ro ­
c h lo r ic a c id th e n b e n to n ite tr e a te d a n d f il te r e d .
B uffer IFEM -3
0.002 M M g C l2.
0.35 M N H 4 C l
0.010 M T ris
0.001 M d ith io th r e ito l (a d d e d j u s t p rio r to u se)
The b u ffe r w a s a d j u s t e d to pH 7.5 a t 2 ° C w ith d i s t i l l e d h y d ro ­
c h lo ric a c id th en b e n to n ite tre a te d and filtered .
25
Buffer LMSW
0.08 M N H 4 Cl
0.0085 M M g(O A c)2
0.01 M T ris
' .
0.01 M p -m e rc a p to e th a n o l (a d d e d j u s t p rio r to u se)
The b u ffe r w a s a d ju s te d to pH 8.1 a t 2 °C w ith d i s t i l l e d h y d ro ­
c h lo r ic a c id th e n b e n to n ite tr e a te d a n d f il te r e d .
B uffer L M SN -16
'0.08 M N H 4 C l
0.016 M M g(O A c)2
0.01 M Tris
0.01 M p -m e rc a p to e th a n o l (a d d e d j u s t p rio r to u se )
The b u ffe r w a s a d ju s te d to pH 7.8 a t 2 °C w ith d i s t i l l e d h y d ro /
c h lo r ic a c id th e n b e n to n ite f ilte r e d .
B uffer MSN
0.08 M N H 4 Cl
0.085 M M g(O A c)2
0.01 M Tris
0.01 M p -m e rc a p to e th a n o l (a d d e d j u s t p rio r to u s e )
The b u ffe r w a s a d j u s t e d to pH 8.1 a t 2 ° C w ith d i s t i l l e d h y d ro ­
c h lo r ic a c i d th e n b e n to n ite t r e a t e d a n d f il te r e d .
26
Buffer N
0.08 M N H 4 C l
0.011 M M g(OAc)2
0.1 M Tris
0.01 M P -m e rc a p to e th a n o l (a d d e d j u s t p rio r to u s e )
The b u ffe r w a s a d ju s te d to pH 8.1 a t 2 °C w ith d i s t i l l e d h y d ro c h lo r­
ic a c id th e n b e n to n ite tr e a te d an d f ilte r e d .
B uffer CA
0.01 M M g(O A c)2
. 0.01 M T ris
The b u ffe r w a s a d ju s te d to pH 7.8 a t 2 °C w ith d i s t i l l e d h y d ro ­
c h lo ric a c id th e n b e n to n ite tr e a te d a n d f il te r e d .
/
B uffer QB
0.5 M N H 4 C l
0.01 M M g(O A c)2
0.01 M Tris
The b u ffe r w a s a d ju s te d to pH 7.8 a t 2 °C w ith d i s t i l l e d h y d ro ­
c h lo ric a c id th e n b e n to n ite tr e a te d a n d f i l t e r e d .
Buffer QC
0.25 M N H 4 Cl
0.01 M M gC l2
0.01 M Tris
27
The b u ffe r w a s a d ju s te d to pH 7.8 a t 2 °C w ith d i s t i l l e d h y d ro ­
c h lo r ic a c id th e n b e n to n ite tr e a te d a n d f ilte r e d .
B uffer P D
0.5 M N H 4 C l
0.01 M M g(O A c)2
0.01 M T ris
The b u ffe r w a s a d ju s te d to pH 7.8 a t 2 °C w ith d i s t i l l e d h y d ro ­
c h lo r ic a c id th e n b e n to n ite tr e a te d an d f il te r e d .
B uffer pH 5'
0.01 M T ris'
0.05 M KCl
0.011 M M g(O A c)2
I
The b u ffe r w a s a d ju s te d to pH 5.2 a t 2 °C w ith d i s t i l l e d h y d ro ­
c h lo r ic a c id th e n b e n to n ite tr e a te d an d f il te r e d .
(d) R e a g e n ts an d ' c h e m ic a ls
A lum ina
L e v ig a te d A b ra siv e G ra in , o b ta in e d from th e N o rto n C o m p an y ,
C a t. # 2 2 0 4 6 1 .
Ammonium S u lfa te
S p e c ia l e n zy m e g r a d e , o b ta in e d from S c h w a rz /M a n n C h e m ic a ls ,
C a t. # 1 9 4 6 . U se d for s a ltin g o u t in itia tio n f a c t o r s .
28
-
Ammonium S u lfa te
R e a g e n t g r a d e , o b ta in e d from th e B ak er-A d am n so n C h e m ic a ls .
U se d fo r s a ltin g o u t v ir u s .
.A rginine
S p e c ific a c ti v ity 306 m C i/m M . O b ta in e d from A m e rsh a m -S e a rle
C o ., C a t. #CFB 62.
C h lo ra m p h e n ic o l
O b ta in e d a s a g ift from P a r k e , D a v is an d C o m p an y .
L y s in e
C
S p e c ific a c ti v ity 312 m C i/m M . O b ta in e d from A m e rsh a m -S e a rle
C o ., C a t. # CFB 6 9.
M illip o re F ilte r s
HAWP n itr o c e llu lo s e f i l t e r s . O b ta in e d from th e M illip o re C o rp o r­
a tio n .
T ry p sin
TPCK tr e a te d tr y p s in , "B" g r a d e , e s s e n t i a l l y fre e o f c h y m o try p s in . O b ta in e d from C a lb io c h e m ., C a t. # 6 4 8 5 2 .
(e) R ib o n u c le a s e R em oval
R ib o n u c le a s e (RMAase) w a s 're m o v e d from g la s s w a r e b y e ith e r:
a) h e a tin g in a dry o v e n a t 2 4 0 °C fo r 10 h o u rs; or b) a u to c la v in g fo r 20
m in u te s a t 15# p r e s s u r e .
RNAase w a s rem o v ed from p l a s t i c w are by
29
e ith e r: a) a u to c la v in g fo r 20 m in u te s (a u to cla va b le p l a s t i c ; o r b ) t r e a t ­
m e n t w ith 15% h y d ro g en p e ro x id e fo r 15 m in u te s .
!
RNAase w a s rem o v ed from b u ffe r s o lu tio n s by a d d in g 2 d ro p s p e r
l i t e r o f a s ta n d a rd b e n to n ite s u s p e n s io n (D unn & H itc h b o m , 19 65) a f te r
th e b u ffe r w a s p re p a re d . The tr e a te d b u ffe r w a s f ilte r e d th ro u g h 'a
M illip o re f iltr a tio n a p p a r a tu s e q u ip p e d w ith a HAWP 047 M illip o re filte r,
a n d c o lle c te d in a s te r ile P y rex b o ttle u s in g s te r ile f il tr a tio n .
(f)
G row th o f p h a g e MS2 on E s c h e r ic h ia c o ll
The fo llo w in g p ro c e d u re p ro d u c e d 15 lite r s o f MS2 v iru s ly s a t e a t
a t i t e r o f a p p ro x im a te ly IO ^ v iru s p e r m l., an d u s e d e s s e n t i a l l y th e
m eth o d o f C la rk (1 9 6 5 ). The i n i t i a l v iru s s o u rc e w a s a ly o p h y liz e d
p o w d e r o f MS2 o b ta in e d from th e A m erican Type C u ltu re -A s s o c ia tio n .
A lo o p o f E. c o li (or o th e r f4" h o s t) w a s .'in o c u la te d from a fre s h
s l a n t in to tw o m l. o f TYS+ b r o th . T his c u ltu re w a s th e n grow n a t 3 7 °C
O
to e a r ly lo g p h a s e , a b o u t 10 b a c te r ia p e r m l., o r u n til p a r tic le s o f
g ro w th c o u ld b e s e e n w ith s w irlin g . O ne drop o f th e re h y d ra te d
A m erican Type C u ltu re A s s o c ia tio n MS2 v iru s w a s th e n a d d e d an d th e
in c u b a tio n c o n tin u e d a t 3 7 °C for s ix to tw e lv e h o u r s . The t i t e r a t th e
e n d o f th is p e rio d w a s a lw a y s a m inim um o f 10
11
v iru s p e r m l. T his
tw o - m l. l y s a t e o f MS2 v iru s w a s n e x t u s e d to in f e c t 100 m l. o f e a r ly lo g
p h a s e E. c o li (in TYS+ ) a t a m u ltip lic ity o f in fe c tio n ( m . o . i . ) o f 8 ,
y ie ld in g a 1 0 0 -m l. l y s a t e a t th e sa m e t i t e r o f 10
11
v iru s p e r m l.
/
30
T w elv e t w o - l i t e r f l a s k s , e a c h c o n ta in in g o n e an d o n e - h a lf l i t e r s
o f TYS+ b r o th , w e re a llo w e d to in c u b a te a t 3 7 °C fo r s ix .to te n h o u r s .
Any f la s k s sh o w in g c lo u d y c o n ta m in a n ts w e re d is c a r d e d . S u ffic ie n t.
E . c o li (from a s to c k s ta tio n a r y p h a s e s o lu tio n ) w a s a d d e d to th e r e m a id in g s t e r i l e f la s k s to y ie ld a c o n c e n tra tio n o f a b o u t 10
6
x
b a c te r ia p e r
m l. The b a c te r ia w e re th e n a llo w e d to gro w to e a r ly lo g p h a s e , a t
w h ic h tim e th e y w e re in f e c te d w ith a n a liq u o t from th e 100 m l. MS2
l y s a t e a t a m . o . i . o f 8 . The f la s k s w e re s w irle d in a G y ro ta ry sS h a k e r
(N ew B ru n sw ick S c ie n tif ic C om pany) a t 370C fo r s ix to e ig h t h o u r s ,
a f te r w h ic h tim e th e y w e re p la c e d in th e c o ld room fo r u s e a b o u t e ig h t
h o u rs l a t e r . The f in a l t i t e r in a ll c a s e s w a s h ig h e r th a n 10
11
v iru s
p e r m l.
The l y s a t e w a s n e x t sp u n in th e GSA ro to r o f th e S e rv a ll c e n tr i­
fu g e fo r 30 m in u te s a t 5,000 RPM (4 0 8 0 x g ) . The s u p e r n a ta n t w a s p o u re d
in to a c h ille d 1 5 - lite r c a rb o y an d m ore l y s a t e w a s a d d e d to th e c e n tr i­
fu g e b o t t l e s , c o lle c tin g th e c e ll d e b ris in a s in g le p e l l e t . Both th e
-carboy an d c e n trifu g e b o ttle s w e re th o ro u g h ly w a s h e d a n d r in s e d w ith
d i s t i l l e d w a te r p rio r to u s e , b u t w e re n o t m ade r ib o n u c le a s e fr e e .
j
A fter c e n trifu g in g th e e n tire 15 l i t e r s , 311 g . o f c r y s ta ll in e re a g e n t
g ra d e am m onium s u lf a te w ere a d d e d p e r l i t e r o f l y s a t e s u p e r n a ta n t and
th e s o lu tio n s tir r e d w ith a m o to riz e d T e flo n p ro p e llo r. The r e s u ltin g
s o lu tio n w a s c h ille d in th e c o ld room fo r e ig h t to te n h o u r s .
31
The p r e c ip ita te w a s re s u s p e n d e d by s tirrin g an d s ip h o n e d in to
GSA ro to r b o t t l e s .- T h e p h a g e p a s te w a s c o lle c te d by c e n tr ifu g a tio n a t
5,000 RPM (4080 xg) fo r 30 m in u te s . N in e m l. o f g la s s d i s t i l l e d w a te r ,
a d ju s t e d to pH 9 w ith s o lid N a O H , w e re a d d e d to e a c h GSA b o t t l e . The
b o ttle s w e re th e n t i l t e d to a llo w th e w a te r to c o n ta c t th e p a s t e an d l e f t
fo r e ig h t to te n h o u rs in th e c o ld room a t 2 ° C .
The s u s p e n s io n w a s th e n c o lle c te d in to a s in g le GSA b o ttle ,
r in s in g th e re m a in in g b o ttle s w ith pH 9 d i s t i l l e d w a te r . F ifty m ic ro ­
gram s o f D N A ase I w a s th e n a d d e d a n d th e en zy m e a llo w e d to w ork u n til
th e p a s t e w a s fre e o f s tr a n d s o f DNA, u s u a lly a b o u t te n m in u te s l a t e r .
' The D N A ase d i g e s t w a s c e n trifu g e d fo r 20 m in u te s a t 13,000 xg in
th e B eckm an M o d el L -2 c e n trifu g e a n d th e p r e c ip ita te d is c a r d e d . The
s u p e r n a ta n t w a s n e x t c e n trifu g e d fo r 20 m in u te s a t 26,000 x g an d th e p r e ­
c ip ita te a g a in d is c a r d e d . The p h a g e w a s now p e lle te d by c e n tr ifu g a tio n
o f th e s u p e r n a ta n t in th e T i-5 0 ro to r for th re e h o u rs a t 60,000 xg in r ib o n u c le a s e fre e c e n tr ifu g e t u b e s .
O ne m l. o f pH 9 d i s t i l l e d w a te r w a s a d d e d to th e b ro w n ish p e lle t
a n d a llo w e d to d is s o lv e o v e rn ig h t. The s o lu tio n w a s c la r if ie d by c e n tr i­
fu g a tio n a t 10,000 xg fo r 15 m in u te s in th e T i-5 0 ro to r . The re s u ltin g
p h a g e s o lu tio n w a s th e n s to re d a t -9 0 °C in th e R evco C o ld C h e s t in
o n e - m l. p o r ti o n s . S u ch a liq u o ts w e re s u ita b le for e x tr a c tio n o f th e
a c tiv e MS2 RNA o v e r a y e a r l a t e r .
32
The v iru s s u s p e n s io n so p ro d u c e d w a s s lig h tly y e llo w an d o p a l­
e s c e n t . It w a s e x tr a c te d d ir e c tly fo r h ig h ly p u rifie d MS2 RNA. For th e
e x tr a c tio n o f h ig h ly p u rifie d MS2 c o a t p r o t e i n , i t w a s r e p e lle te d tw ic e
a t 60,000 xg fo r th r e e h o u rs to o b ta in a c le a r p e l l e t .
The v iru s s u s p e n s io n h a d th e u ltr a v io le t sp e c tru m a s sh o w n in
F ig . 4 a n d a n e x tin c tio n c o e f f ic ie n t o f o n e o p tic a l d e n s ity u n it p e r m g.
p e r m l. T h e se p h y s ic a l c o n s ta n ts ' a g re e d w ith th o s e o f S tr a u s s &
S ih s h e im e r (1 9 6 3 ). S e d im e n ta tio n v e lo c ity p ro f ile s o b ta in e d in th e
B eckm an M o d el E a n a ly t ic a l u ltr a c e n tr if u g e y ie ld e d a s in g le p e a k o f
80 S v e d b e rg u n i t s . E le c tro n m ic ro g ra p h s m ade by th e a u th o r o f th e p u ri­
fie d v iru s sh o w e d p o ly h e d ra l b o d ie s o f a p p ro x im a te ly 30 m p w h ic h w ere
p h o to g ra p h ic a lly id e n tic a l to e le c tr o n m ic ro g ra p h s ta k e n o f th e MS2
v iru s b y S tra u s s & S in s h e im e r (1 9 6 3 ). F,ig. 5 d e p ic ts a flo w c h a r t on th e
g ro w th an d p u rif ic a tio n o f MS2 v i r u s .
(g) E x tra c tio n o f m e s s e n g e r RNA from p h a g e MS2
The fo llo w in g p ro c e d u re u t i l i z e s th e fro z e n p h a g e s u s p e n s io n .
The RNA p ro d u c t o b ta in e d i s fu lly c a p a b le o f d ir e c tin g in v itro p ro te in
s y n th e s is in c o m b in a tio n w ith E. c o li rib o s o m e s an d s u p e r n a ta n t
fa c to rs .
O ne m l. o f a th a w e d p h a g e s u s p e n s io n w a s a d d e d to a rib o n u c l e a s e fre e 12- m l. c o n ic a l c e n trifu g e tu b e . O ne m l. o f g l a s s d is tille d
w a te r s a tu r a te d p h e n o l ( r e d is tille d r e a g e n t g rad e) a n d 0.1 m l. o f 2 M
33
am m onium c a r b o n a te (in g l a s s d i s t i l l e d w a ter) w e re a d d e d s im u lta n e ­
o u s ly . The s u s p e n s io n w a s g e n tly m ix e d w ith a r ib o n u c le a s e free
P a s te u r p ip e tte for te n m in u te s a t room te m p e r a tu r e , ta k in g c a re to a v o id
f r o th in g . The m ix tu re a t th is p o in t h a d a c lo u d y lig h t bro w n c o lo r.
The s o lu tio n w a s th e n c e n tr ifu g e d a t fu ll s p e e d in th e c lin ic a l
c e n tr ifu g e a t 2° C fo r 15 m in u te s . The to p a q u e o u s la y e r c o n ta in in g th e
RNA w a s re m o v e d w ith a r ib o n u c le a s e fre e P a s te u r p ip e tte a n d p la c e d
in to a s e c o n d c h ille d r ib o n u c le a s e fre e c o n ic a l c e n tr ifu g e tu b e . T his
a q u e o u s e x tr a c t u s u a ll y tu rn e d c lo u d y w h ite w ith in a fe w m in u te s .
O ne m l. o f c o ld g l a s s d i s t i l l e d w a te r w a s -th e n a d d e d to th e
p h e n o l p h a s e fo r a n a d d itio n a l e x tr a c tio n o f RNA. The s o lu tio n w as
m ix e d a n d c e n tr ifu g e d a s a b o v e a n d th e to p a q u e o u s la y e r a d d e d to th e
f i r s t a q u e o u s e x tr a c t. The c o a t p ro te in c o n ta in in g p h e n o l p h a s e w a s
s a v e d fo r fu rth e r p u r if ic a tio n .
All t r a c e s o f p h e n o l w e re re m o v e d from th e a q u e o u s la y e r b y e x ­
tr a c tin g s ix tim e s w ith r e a g e n t g ra d e e th e r in th e c o ld ro o m . The c e n ­
trifu g e tu b e w a s f ille d to w ith in o n e c e n tim e te r o f th e to p w ith e th e r
a n d e x tr a c te d by m ix in g w ith a P a s te u r p i p e t t e . The c o n ic a l c e n trifu g e
tu b e w a s c e n tr ifu g e d a t to p s p e e d in th e c lin ic a l c e n tr ifu g e fo r 15 m in ­
u te s a n d th e e th e r la y e r a s p ir a te d a n d d is c a r d e d . At th e c o m p le tio n o f
th e s ix th e x tr a c tio n th e bottom a q u e o u s p h a s e w a s c l e a r .
All t r a c e s o f e th e r w e re re m o v e d b y g e n tly b lo w in g a j e t o f n itr o ­
g e n o v e r th e s o lu tio n (in a n ic e b a th ) u n til a ll o d o r o f e th e r w a s g o n e .
34
S ix d ro p s o f c o ld 3 M so d iu m a c e ta te (in g l a s s d i s t i l l e d w a te r) w ere
th e n a d d e d an d th e s o lu tio n m ix e d . The f in a l flo c c u la tio n o f RNA w a s
a c h ie v e d b y th e a d d itio n o f tw o v o lu m e s o f c o ld r e a g e n t g ra d e 100%
e th a n o l an d m ix in g . If th e p r e c ip ita te re m a in e d d i s p e r s e d , SM sodium
a c e t a t e w a s a d d e d d ro p w is e u n til th e s o lu tio n b e g a n to f lo c c u la te . In
m o s t c a s e s no e x tra so d iu m a c e ta te w a s re q u ire d .
The f lo c c u la te d RNA s o lu tio n w a s a llo w e d to s i t fo r 40 m in u te s ,
fo llo w e d by c e n tr ifu g a tio n in th e c lin ic a l c e n trifu g e fo r 20 m in u te s a t
to p s p e e d in th e c o ld room . The e th a n o l w a s d e c a n te d , a n d th e p a s te
ly o p h y liz e d to a c r u s ty w h ite d r y n e s s . Two h u n d red fifty to fiv e h u n ­
d re d m ic r o lite r s o f c o ld g l a s s d i s t i l l e d w a te r w a s a d d e d to th e d ry RNA.
a n d a llo w e d to d is s o lv e o v e rn ig h t.
The fo llo w in g d a y th e u ltr a v io le t s p e c tru m o f th e p ro d u c t w as o b ­
ta in e d to d e te rm in e th e c o n c e n tra tio n o f RNA. An e x tin c tio n c o e f f ic ie n t
o f 25 o p tic a l d e n s ity u n its p e r m g. o f RNA p e r m l. o f w a te r w a s u s e d ,
a n d th e RNA b o ttle d in o n e -m g . p o r ti o n s . S u ch a liq u o ts w e re s to re d a t
-9.0°C in th e R evco C o ld C h e s t u n til u s e d . A c tiv ity h a s b e e n found to
d e c r e a s e a p p ro x im a te ly 50% o v e r a tw e lv e -m o n th p e r io d , a s m e a su re d
by i t s s tim u la tio n o f ^ C - a m in o a c id in c o rp o ra tio n . F ig . 6 d e p ic ts a
flo w d ia g ra m o f th e e x tr a c tio n a n d p u r if ic a tio n o f MS2 RNA.
4
35
(h)
E x tra c tio n o f p h a g e M S2 c o a t p ro te in
T his -p ro ced u re u s e d a p h a g e s u s p e n s io n w h ic h h a d b e e n fu rth e r
p u rifie d by p e lle tin g th e v iru s tw ic e a t 93,000 x g for 3 h o u r s . The p e lle t
th e n a p p e a re d c le a r a n d g a v e a s in g le s h a rp p e a k o f 80s in th e B eckm an
M o d el E a n a ly t ic a l u ltr a c e n tr if u g e .
Ten v o lu m e s o f c o ld r e d i s t i l l e d r e a g e n t g ra d e a c e to n e w e re a d d e d .
to th e p h e n o l p h a s e p ro d u c e d by a n RNA e x tr a c tio n o f th e v i r u s . The
m ix tu re w a s a llo w e d to s i t for '20 m in u te s a t 2 °C fo llo w e d b y c e n tr ifu g a ­
tio n a t to p s p e e d in th e c lin ic a l c e n tr ifu g e fo r 45 m in u te s . The r e s u l t ­
in g s u p e r n a ta n t w a s a s p ir a te d an d d is c a r d e d w h ile th e p e l l e t w a s
w a s h e d tw ic e w ith 5 m l. o f c o ld d i s t i l l e d a c e to n e by r e s u s p e n s io n an d
c e n tr ifu g a tio n for 20 m in u te s a t to p sp e e d , in th e sam e c e n tr if u g e .
The w a s h e d p ro te in p e l l e t w a s s u s p e n d e d in o n e m l. o f 0.5 M
so d iu m h y d ro x id e a n d d ia ly z e d a t 2 °C a g a in s t o n e l i t e r o f 0.5 M sodium
h y d ro x id e u s in g a fo rc e d flo w d ia ly z e r . W hen p e rfo rm ic a c id o x id a tio n
w a s p la n n e d , th e p ro te in w a s d ia ly z e d a g a in s t d i s t i l l e d w a te r a t 2 °C to
rem ove a ll tr a c e s o f so d iu m h y d ro x id e .
U sin g th e e x tin c tio n c o e f f ic ie n t o f o n e o p tic a l d e n s ity u n it per mg.
per ml. of water (S tra u s s & S in s h e im e r , 1962), th e p ro te in w a s ly o p iliz e d to
d ry n e s s a n d s to re d in 1.0-m g. p o rtio n s in th e R evco C o ld C h e s t a t
-9 0 °C .
36
(i) P re p a ra tio n o f S -3 0 e x tr a c ts
The fo llo w in g m eth o d u tiliz e d E s c h e r ic h ia c o li Q-13 w h ic h is a
n itro g e n m u s ta rd in d u c e d m u ta n t d e f ic ie n t in c e ll u la r r ib o n u c le a s e s ,
th u s p e rm ittin g th e p re p a ra tio n o f S -3 0 e x tr a c ts lo w in t h e s e rib o n u ­
c le a s e s .
The e x tr a c tio n h a s a ls o b e e n s u c c e s s f u lly u s e d on Jjb c o li
M R E-600 a n d K -12. The m eth o d fo llo w s t h a t o f C la rk (19 6 5 ).
T w enty gram s o f fro z e n E. c o li c e ll s w e re p la c e d in a la rg e m o rta r
m a in ta in e d a t 2 ° C , a n d to i t a d d e d 10 g . o f a lu m in a . G rin d in g w a s b e ­
gun w ith o u t a llo w in g th e c e ll s to th a w , a n d m ore a lu m in a a d d e d a s
n e c e s s a r y to k e e p th e c e ll p a s te c o v e r e d . A fter fiv e m in u te s o f g rin d ­
in g , 5 m l. o f c o ld b u f f e r N w a s a d d e d a n d g rin d in g c o n tin u e d fo r a n o th e r
fiv e m in u te s . A to ta l o f 40 m l. o f b u f f e r N an d 30 g . o f a lu m in a w ere
a d d e d d u rin g th e g rin d in g p r o c e d u r e .
The g round c e ll s w e re tr a n s f e r r e d to a r ib o n u c le a s e fre e 2 5 0 -m l.
b e a k e r , th e c e l l s a llo w e d to th a w c o m p le te ly an d 20 tag. o f D N A ase I
a d d e d from a s to c k s o lu tio n o f o n e m g. D N A ase p e r m l. o f g la s s d i s ­
til l e d w a te r . The p a s te w a s th e n g e n tly s tir r e d w ith a g l a s s rod u n til
th e D N A ase h a d c o m p le te d i t s a c t i o n , u s u a lly w ith in fiv e m in u te s .
The c e ll p a s te w a s tr a n s f e r r e d to c le a n 3 0 -m l. p o ly c a rb o n a te c e n ­
trifu g e tu b e s a n d c e n trifu g e d a t 2 ° C fo r 10 m in u te s a t 10,000 xg in th e
B eckm an M o d el L ty p e 30 ro to r . The s u p e r n a ta n t w a s th e n tra n s fe rre d to
c le a n 3 0 -m l. p o ly c a r b o n a te tu b e s a n d sp u n for 30 m in u te s a t 30,000 xg
a t 2 °C in th e sa m e r o t o r , d is c a rd in g th e p r e c ip ita te . The to p
/
.
th r e e - f o u r th s o f th is s u p e r n a ta n t (th e S -3 0 fra c tio n ) w a s rem o v ed w ith a
r ib o n u c le a s e fre e s y rin g e a n d tr a n s f e r r e d to a r ib o n u c le a s e fre e 2 5 0 -m l.
b e a k e r . The b ottom o n e -fo u rth o f th e s u p e r n a ta n t a n d p r e c ip ita te w as
d is c a r d e d .
.
.
In som e c a s e s a F re n c h P r e s s u r e C e ll w a s u s e d to ly s e th e c e lls
( s e e " M e th o d s: P re p a ra tio n o f c ru d e in itia tio n facto rs")* . The e n s u in g
s t e p s , h o w e v e r, w e re id e n tic a l to th o s e fo llo w in g th e a lu m in a g rin d in g .
The S -3 0 fr a c tio n w a s s u b s e q u e n tly 'p r e - in c u b a te d u n d e r c o n d i­
tio n s w h ic h p e rm itte d p ro te in s y n th e s is to o c c u r an d a llo w e d a n y free
e n d o g e n o u s mRNA a n d tRNA to b e d is c h a r g e d from th e rib o s o m e s . To
e a c h 16.0 m l. o f th e S -3 0 e x tr a c t th e fo llo w in g w e re a d d ed :
2.0 m l. o f 0.12 M PEP (in b u f f e r N o r LMSN)
1.0 m l. o f 0.05 M ATP (in b u f f e r N o r LMSN)
0.6 m l. o f 0.01 M GTP (in b u ffe r N o r LMSN)
-3
0.8 m l. o f a 10 M s o lu tio n o f e a c h o f th e 20 L a m in o a c id s in
b u ffe r N
30 EU o f p y ru v a te k in a s e
The s o lu tio n w a s m ixed g e n tly a n d in c u b a te d a t 3 7 °C fo r 40 m in u n te s . D ia ly s is th e n e n s u e d in c e ll u lo s e tu b in g w h ic h h a d b e e n p r e -3
tr e a te d by a u to c la v in g for 15 m in u te s in 10 M NaEDTA a n d 1% N aH C O g
fo llo w e d by e x te n s iv e r in s e s w ith d i s t i l l e d w a te r .
The S -3 0 fr a c tio n w a s d ia ly z e d fo r 12 h o u rs a t 2 °C a g a i n s t th re e
e q u a lly s p a c e d c h a n g e s o f tw o l i t e r s o f b u ffe r N (or LMSN) . Upon
38
c o m p le tio n o f d ia l y s i s th e s o lu tio n w a s tr a n s f e r r e d to c h ille d 3 0 -m l.
p o ly c a r b o n a te c e n trifu g e tu b e s an d c la r if ie d a t 20,000 x g fo r 10 m in u te s
a t 2 ° C in th e B eckm an M o d el L ty p e 30 r o to r . The to p th r e e - f o u r th s o f
th e s o lu tio n w a s th e n p la c e d in to r ib o n u c le a s e free v i a l s , 1.0 m l. p e r
v i a l , a n d s to re d in th e R evco C o ld C h e s t a t - 9 0 ° C .
The s t a b i l i t y o f p re p a ra tio n s s o p re p a re d sh o w e d an a c tiv ity lo s s
o f l e s s th a n 50% a f te r 14 m o n th s o f s to ra g e w h en a s s a y e d a g a in s t an
MS2 RNA d ir e c te d in v itro s y s te m .
(j) R ibosom e p re p a ra tio n
All r e a g e n t s , g l a s s w a r e , c e n tr if u g e t u b e s , a n d m a te r ia ls to be
u s e d in th e p re p a ra tio n w e re m ade s t e r i l e a n d RNAase fre e b y th e a p p ro ­
p r ia te tr e a tm e n t ( s e e " M a te r ia ls a n d M e th o d s: R ib o n u c le a s e re m o v a l").
The p ro c e d u re fo llo w e d th a t o f S ta n le y , S a l a s , W ah b a & O c h o a (1 9 6 6 ).
Two h u n d re d g ram s o f fro z e n E s c h e r ic h ia c o li s tr a in Q-13 or
M RE-600 w e re g ro u n d in th r e e p o rtio n s on a la rg e m o rtar an d p e s tle
u n til th e c e ll s w e re s lig h tly th a w e d . A p p ro x im a te ly 70 gm . o f a lu m in a
w e re a d d e d to e a c h p o rtio n a n d th e m ix tu re g ro u n d to a th ic k p a s t e .
A p p ro x im a te ly 35 m l. o f b u ffe r N w e re a d d e d to e a c h p o rtio n a n d th e m ix ­
tu re g ro u n d to a s lu r r y . T his p ro c e d u re y ie ld e d a p p ro x im a te ly 350 m l.
t o t a l v o lu m e .
The g round h o m o g e n a te w a s th e n p la c e d in c e n tr ifu g e tu b e s an d
c e n tr ifu g e d 40 m in u te s a t 15,000 RPM (39,000 xg) in th e S S -3 4 ro to r o f th e
S o rv a ll RC2-B c e n trifu g e to rem o v e th e a lu m in a and c e ll d e b r is . The
39
s u p e r n a ta n t fra c tio n w a s rem ov ed an d 0.6 m g. o f d e o x y rib o n u c le a s e I
(W o rth in g to n B io c h e m ic a ls) w a s a d d e d an d s tir r e d in to th is f r a c tio n .
The s u p e r n a ta n t fr a c tio n w a s k e p t a t 4 ° C fo r 90 m in u te s to a llo w co m ­
p le te d ig e s tio n o f th e DNA. The D N A ase tr e a te d fr a c tio n w a s th e n c e n ­
trifu g e d in th e 30 ro to r o f th e B eckm an M o d el L-2 u ltr a c e n trifu g e a t
18,000 RPM (30,000 xg) fo r tw o h o u rs . The s u p e r n a ta n t fr a c tio n w a s r e ­
m oved a n d tr a n s f e r r e d to tu b es s u ita b le , fo r th e 5 0 -T i r o t o r , th e n c e n tr i­
fu g e d fo r 4 h o u rs a t 50,000 RPM (170,000 xg) in th e M o d el L -2 u ltr a c e n ­
tr if u g e . The to p 90% o f th e s u p e r n a ta n t fr a c tio n w a s rem o v ed w ith a
s y rin g e a n d s a v e d a s S - 1 0 0 . F iv e m illilite r s o f b u f f e r O C + w e re a d d e d
to e a c h tu b e a n d th e p e l l e t a llo w e d to d is s o lv e o v e rn ig h t w ith o c c a ­
s io n a l s tirrin g o f th e p e l l e t s u rfa c e w ith a s tirrin g ro d .
.The tu b e s c o n ta in in g th e d is s o lv e d p e l l e t w e re th e n f ille d w ith
b u ffe r O C+ an d c e n trifu g e d for 15 m in u te s a t 25,000 RPM (42,000 xg) in th e
ty p e 5 0 -T i ro to r to rem ove e x tra n e o u s d e b r is . The s u p e r n a ta n t fra c tio n
w a s th e n la y e r e d on a d ie th y la m in o e th y l (DEAE)- c e l l u l o s e colum n p r e v i­
o u s ly p re p a re d ( s e e " M a te r ia ls an d M e th o d s : DEAE co lu m n p re p a ra tio n " )
a n d th e colum n w a s h e d w ith 2 l i t e r s o f b u ffe r O C +. The rib o s o m e s w ere
e lu te d from th e colum n .w ith b u ffe r OD+ an d c o n c e n tra te d b y c e n tr ifu g a ­
tio n a t 50,000 RPM fo r 4 h o u rs in th e 5 0 -T i ro to r o f th e M o d e l L -2 .
The .rib o so m e s w e re d is s o lv e d in a s m a ll am o u n t o f b u ffe r BC.
The c o n c e n tra tio n o f rib o s o m e s w a s d e te rm in e d by m e a s u rin g th e ir a b ­
s o rp tio n a t 2 60 mp w h ic h th e n p e rm itte d th e c o n c e n tra tio n to be
40
a d ju s t e d to e ith e r 2 5 o r 50 m g ./m l. The r e s u ltin g s o lu tio n w a s th e n
b o ttle d in s m a ll a liq u o ts (0.25 to 0.5 m l.) a n d k e p t fro z e n a t -9 0 ° C
p rio r to u s e .
(k)
DEAE C olum n p re p a ra tio n — p u rifie d rib o s o m e s
Two h u n d re d g ram s o f DEAE c e ll u lo s e (C a lb io c h e m ) w e re w a s h e d
w ith 2 l i t e r s o f 0.1 N so d iu m h y d ro x id e b y s u c tio n f i l t r a t i o n , w a s h e d to
n e u tr a lity w ith d is tille d , w a t e r , w a s h e d w ith 2 l i t e r s 95% e t h a n o l , an d
re w a s h e d w ith d i s t i l l e d w a te r . The r e s in w a s th e n s u s p e n d e d in d i s ­
t i l l e d w a te r and- a u to c la v e d fo r 20 m in u te s . The r e s in w a s p o u re d in to a
T y p e K S -1 0 0 P h a rm a c ia colum n (5.0 x 100 c m . ) a n d p a c k e d u n d e r IO p si
n itr o g e n p r e s s u r e . The colum n w a s th e n w a s h e d w ith 4 l i t e r s o f b u ffe r
O C +. The fin a l d im e n s io n s o f th e r e s in w e re 61.x5 cm .
. (I ) P re p a ra tio n o f pH 5' fra c tio n
A co lu m n w a s p re p a re d by a u to c la v in g 50 g . o f S e p h a d e x G -5 0 •
c o a r s e in b u ffe r pH 5' fo r o n e - h a lf h o u r. A fter c o o lin g to room te m p e ra ­
tu re , th e " f in e s " w e re a s p ir a te d o ff. The r e s in w a s re s u s p e n d e d
fo llo w e d b y a s p ir a tio n o f th e " f in e s " tw o a d d itio n a l tim e s . F in a lly , th e
r e s in w a s s u s p e n d e d in b u ffe r pH 5' a n d p o u re d in to a 2 c m .x l 7 cm,.
P y rex c o lu m n 'e q u ip p e d w ith a n e x tra c o u rs e p o ro s ity s in te r e d g la s s
d i s c o n to w h ic h th e r e s in s e t t l e d . A s to p c o c k g o v e rn e d th e flow r a t e „
The co lu m n w a s th e n w a s h e d w ith 100 m l. o f b u ffe r pH 5' a n d y ie ld e d a
fin a l r e s in s iz e o f 2 cm. x 12 c m .
41
The pH 5' fr a c tio n w a s p re p a re d b y lo a d in g th e co lu m n w ith 1.0 to
2.0 m l. o f th e S-100 fr a c tio n an d e lu tin g w ith b u ffe r pH 5 '. The m ilky
w h ite p r e c ip ita te w a s c o lle c te d in a r ib o n u c le a s e fre e 1 5 -m l. c o n ic a l
c e n tr ifu g e t u b e , u s u a lly a b o u t 6.0 m l. The p r e c ip ita te w a s th e n c e n tr i­
fu g e d a t to p s p e e d in th e c lin ic a l c e n trifu g e fo r 15 m in u te s . The s u p e r ­
n a ta n t w a s d is c a r d e d a n d th e p r e c ip ita te w a s h e d tw ic e w ith b u ffe r pH 5'
by r e s u s p e n s io n a n d c e n tr if u g a tio n . The p r e c ip ita te w a s f in a lly d i s solv- - in 0.25 to 1.0 m l. o f b u fferL M S N -1 6 a n d u s e d a s th e pH 5' f r a c ­
tio n . In c a s e s w h e re som e s e d im e n t re m a in e d a f te r d is s o lv in g in b u ffe r
LM SN -16, i t w a s re m o v e d b y c e n tr ifu g a tio n a t to p s p e e d in th e c lin ic a l
c e n trifu g e for 10 m in u te s .
(m) P re p a ra tio n o f c ru d e in itia tio n fa c to rs
S e v e ra l m e th o d s w e re tr ie d in th e p u r if ic a tio n o f c ru d e in itia tio n
f a c t o r s . The p o ly e th y le n e g ly c o l/d e x tr a n s e p a r a tio n re p o rte d by
.H e rz b e rg , b e lo n g & R ev el (19 69) p ro v e d to b e b o th tim e c o n su m in g and
y ie ld e d a lo w re c o v e ry . As a r e s u l t , th e p ro c e d u re s d e v e lo p e d by
W a h b a , C h a e , I w a s a k i , M a x u m d e r, M ille r , e t a l . (19 69) w e re u tiliz e d .
T his m eth o d in v o lv e d w a s h in g a rib o s o m a l p e lle t w ith a h ig h io n ic
s tr e n g th b u f f e r , an d re c o v e rin g th e c ru d e fa c to rs by a n am m onium s u l ­
fa te p r e c ip ita tio n .
I n itia tio n fa c to rs w e re p re p a re d from E s c h e r ic h ia c o li MRE-600
.w h ich h a d b e e n grow n on LS m edium to e a r ly lo g p h a s e . The b a c te r ia
w e re th e n w a s h e d tw ic e w ith 0.2 M N H ^C l an d o n c e w ith b u ffe r N . They
42
w e re th e n d iv id e d in to 2 5 - g . p o rtio n s a n d s to re d a t - 9 0 °C in th e R evco
C o ld C h e s t.
To 25 g . o f f r e s h ly th a w e d a n d p a c k e d ER c o li M RE-600 w ere
a d d e d 2 5 m l. o f b u f f e r . The m ix tu re w a s s tirre d an d im m e d ia te ly
p a s s e d th ro u g h th e F re n c h P r e s s u r e C e ll a t a p r e s s u r e o f b e tw e e n 10,000
a n d 25,000 p s i . The l y s a t e w a s c o lle c te d in to a r ib o n u c le a s e free
2 5 0 -m l. c h ille d b e a k e r . Two h u n d re d m ic ro g ra m s o f D N A ase w a s th e n
a d d e d a n d th e en zy m e a llo w e d to c o m p le te i t s a c ti o n , u s u a lly w ith in
fiv e m in u te s . The s u s p e n s io n w a s th e n p o u re d in to B eckm an 3 0 -m l.
p o ly c a rb o n a te c e n trifu g e tu b e s a n d sp u n a t 18,000 RPM (30,000 xg) for 30
m in u te s in th e M o d el L -2 p re p a ra tiv e u ltr a c e n trifu g e a t 2 0 C . The p e lle t
w a s d is c a r d e d a n d th e u p p e r th r e e - f o u r th s o f th e s u p e r n a ta n t w a s sp u n
a g a in for 4 h o u rs a t 50,000 RPM (93,000 xg) in th e T i-5 0 ro to r u s in g 10-ml.
p o ly c a r b o n a te s c re w c a p c e n trifu g e t u b e s . The to p tw o - th ir d s o f th is
s u p e r n a ta n t w a s c a r e f u lly rem o v ed a n d b o ttle d in 1 .0 -m l. p o r t i o n s , d e s ­
ig n a te d IFS-100 (th e S-100 fra c tio n o f an in ita tio n f a c to r p r e p a r a tio n ) .
The re m a in in g o n e - th ir d o f th e s u p e r n a ta n t w a s d is c a r d e d .
To th e p e l l e t w a s a d d e d a p p ro x im a te ly tw ic e th e v o lu m e o f b u ffe r
Bjf2 • The p e l l e t w a s th e n g e n tly b u t c o m p le te ly s t i r r e d , a n d th e n
a llo w e d to s o a k o v e rn ig h t.
U pon c o m p le tio n o f th is rib o s o m a l h ig h s a l t w a s h , th e s o lu tio n
w a s c la r if ie d by c e n tr ifu g a tio n a t 2 °C fo r 30 m in u te s a t 20,000 RPM
(3 0 ,0 0 0 xg) in th e T i-5 0 r o to r i The s u p e r n a ta n t from th is s p in w a s
f in a lly c e n tr ifu g e d a t 2 °C fo r 4 h o u rs a t 50,000 RPM (93,000 xg), a g a in in
th e T i-5 0 ro to r . The s u p e r n a ta n t from th is s p in c o n ta in e d th e cru d e
in itia tio n f a c t o r s , a lo n g w ith o th e r m o le c u le s w a s h e d o ff th e rib o s o m e s
by th e h ig h s a l t w a s h .
T his s u p e r n a ta n t w a s m ad e 35% s a tu r a te d (a t 2 °C ) w ith en zy m e
g ra d e am m onium s u lf a te b y th e a p p ro p ria te am o u n t o f b u ffe r A g fg . A fter
a llo w in g f lo c c u la tio n to o c c u r fo r 40 m in u te s , th e p r e c ip ita te w a s c o l­
l e c t e d by c e n tr ifu g a tio n in th e 8 8 -3 4 ro to r o f th e S e rv a ll c e n trifu g e fo r
15 m in u te s a t 15,000 RPM (22,000 x g ). The p r e c ip ita te w a s d is c a r d e d .
To th e a b o v e 35% s a tu r a te d s u p e r n a ta n t w a s a d d e d s u f f ic ie n t
b u ffe r Ac L to b rin g th e am m onium s u lf a te s a tu r a tio n to 75% . The s o lu tio n w a s a g a in a llo w e d to f lo c c u la te fo r 40 m in u te s a n d c e n trifu g e d a s
w a s th e 35% p r e c i p i t a t e . The s u p e r n a ta n t w a s d is c a r d e d a n d th e p r e ­
c i p i t a t e r e ta in e d a s th e s o u rc e o f c ru d e in itia tio n f a c t o r s .
The p e l l e t o f c ru d e in itia tio n f a c to rs w a s r in s e d w ith 75%
am m onium s u lf a te s a tu r a te d b u ffe r Agfg a n d s to re d a t 20 C w ith a v o lu m e
o f 75% s a tu r a te d b u f f e r Agfg s u f f ic ie n t to c o v e r th e p e l l e t .
S uch p r e p a r a tio n s r e ta in e d th e ir s tim u la to ry a c ti v ity for p u rifie d
in v itr o s y s te m s fo r up to th re e w e e k s , a f te r w h ic h tim e th e ir s tim u la ­
to ry a c ti v ity d e c r e a s e d to l e s s th a n 10%.
For in v itro in c o r p o r a tio n s , c ru d e in itia tio n fa c to r s from S g . o f
c e l l s w e re d ia ly z e d on th e fo rc e d flo w d ia ly z e r a g a in s t tw o 1 - lite r
c h a n g e s o f b u ffe r L M SN -16. The u ltr a v io le t s p e c tr a o f th e c le a r
44
s o lu b iliz e d p ro d u c t w a s th e n t a k e n , a n d th e s o lu tio n w a s u s e d im m e­
d ia te ly .
The fin a l c o n c e n tra tio n o f c ru d e in itia tio n fa c to rs w a s u s u a lly
b e tw e e n 5 a n d IOAggQ o p tic a l d e n s ity u n its p e r m l.
(n) In v itr o in c o rp o ra tio n p ro c e d u re s ;.
In c o rp o ra tio n w ith S -3 0 e x tr a c ts
I
I n c u b a tio n s w e re c a r rie d o u t in 12- m l. r ib o n u c le a s e fre e c o n ic a l
c e n tr ifu g e t u b e s . All g l a s s w a r e , p ip e tte s an d m a te ria ls w e re a ls o r ib o ­
n u c le a s e f r e e . O ne o r tw o m illilite r in c u b a tio n v o lu m e s w e re u s e d , d e ­
p e n d in g on th e in te n d e d u s e . The c o n te n ts o f a s ta n d a rd o n e -m l.
in c u b a tio n w e re a s f o ll o w s , a ll r e a g e n ts b e in g m ade up in b u ffe r LMSN:
0.05 m l. o f 0.08 M ATP
0.05 m l. o f S xlO - 4 M GTP a n d CTP
0.05 m l. o f 0.005 M PEP
0.05 m l. o f 30.0 l U / m l . p y ru v a te k in a s e
0.10 m l. o f 4 m g ./m l. E. c o ll tRNA
0.10 m l. o f 10 ia C i/m l.
- I y s in e
0.10 m l. o f 10 p C i/m l. ^ C - a r g in in e
__O
0.05 m l. o f a s o lu tio n 10 M in e a c h L -am in o a c id e x c e p t h i s t i ­
d in e , a rg in in e an d ly s in e w h ic h w ere d e le te d
0.20 m l. o f p r e - in c u b a te d a n d d ia ly z e d S -3 0
0.10 m l. o f I m g ./m l. MS2 RNA
0.15 m l. o f b u f f e r LMSN
45
E a c h o n e - m illi lite r in c o rp o ra tio n th e re fo re ’ c o n ta in e d th e fo llo w in g
in g r e d ie n ts :
4.0 PM ATP
0.02 5 pM GTP an d CTP
0.02 5 PM PEP
5 x 1 0 ^ pM e a c h L -am in o a c id (e x c e p t h i s t i d i n e , a rg in in e and
ly s in e )
■
1.5' IU p y ru v a te k in a s e
1.0 pC i
14
C - I y s in e
1.0 pC i
14
C -a rg in in e
400 p g . E . c o li tRNA
100 p g . MS2 RNA
0.2 m l. S -3 0 fra c tio n
10 PM T ris
80 pM N H 4 Cl
8.5 pM Mg(OAc) g
E ach 12 - m l. in c u b a tio n tu b e w a s s u p p lie d w ith a l l in g re d ie n ts ex^c e p t th e MS2 mRNA. W hen a ll th e tu b e s w e re r e a d y , MS2 mRNA w a s
a d d e d u s in g e ith e r a 1 0 0 -p l. m ic ro p ip e tte o r a lO O -pl. E p p e n d o rf m ic ro p ip e tte r .
The m ix tu re w a s th e n g e n tly a g ita te d u tiliz in g a V ortex Jr
m ix e r an d th e tu b e p la c e d .in a 37c>C w a te r b a th for th e a p p ro p ria te
in te r v a l o f tim e .
46
U pon c o m p le tio n o f th e in c u b a tio n , e ith e r: a) th e ra d io a c tiv e
p o ly p e p tid e p ro d u c ts w e re p r e c ip ita te d w ith tr ic h lo r o a c e tic a c i d , or
b) th e
C - p e p tid e s w e re d ig e s te d by tr y p s in an d fin g e rp r in te d .
C o u n tin g o f to ta l a c id p r e c ip ita b le c o u n t s .
W hlBn c o u n tin g o f
t o t a l a c id p r e c ip ita b le c o u n ts w a s d e s i r e d , o n e m illilite r in c u b a tio n
v o lu m e s w e re e m p lo y e d . At th e te rm in a tio n o f th e 3 7 °C in c u b a tio n ,
0.3 m g. o f b o v in e seru m a lb u n in (I m g ./m l. d i s t i l l e d w a ter) w a s a d d e d a s
a p ro te in c a r r ie r a n d th e s o lu tio n w a s m ix e d . S ix m illi lite r s o f 5% t r i ­
c h lo r o a c e tic a c id w a s th e n a d d e d a n d th e s o lu tio n w a s m ix e d w e l l .
H e a tin g th e m ix tu re to 9 0 °C fo r 15 m in u te s w a s n e x t p e rfo rm ed to h y d ro ­
ly z e th e a c y l bond's o f a m in o a c id s a n d p o ly p e p tid e s e s te r if ie d to tRNA
m o le c u le s . The m ix tu re w a s c o o le d to 2 ° C a n d f ilte r e d th ro u g h M illip o re HAWP 2 .5 -c m . n itr o c e llu lo s e f i l t e r s .
E ach f ilte r e d p r e c ip ita te w a s
s u b s e q u e n tly w a s h e d w ith a t l e a s t 100 m l. o f 5% tr i c h lo r o a c e t ic a c i d , a
v i t a l s te p in re d u c in g b a c k g ro u n d r a d io a c tiv ity b e c a u s e o f th e ^ C - a m in o
a c id s re te n tio n on th e f i l t e r . The f il te r s w e re th e n re m o v e d from th e
M illip o re f iltr a tio n a p p a r a t u s , m o u n te d on a p p ro p ria te c o u n tin g p la n c h e t s , a n d d rie d a t 7 0 °C fo r 15 m in u te s . The s a m p le s w e re c o u n te d in a
N u c le a f - C h ic a g o m odel 4312 3 -c m . w in d o w e d
gas
flo w c o u n te r .
T y p ic a l in c u b a tio n s g a v e w e ll o v e r 12,000 c p m /m l. in c u b a tio n ,
w ith a b a c k g ro u n d o f l e s s th a n 150 cp m .
I
47
F in g e rp rin tin g o f th e
P a rt I:
14
C in v itro p e p tid e s .
P re p a ra tio n o f th e
W hen fin g e rp rin tin g o f
14
C -p ro d u c ts for tr y p tic d ig e s tio n .
14
'
■
C in v itro p e p tid e s w a s d e s i r e d , 2 -m l. in c o r­
p o ra tio n v o lu m e s w e re e m p lo y e d . In a d d itio n , th e a m o u n t o f MS2 J11RNA
a d d e d w a s c a r e f u lly a d ju s te d to g iv e a t l e a s t 50,000 a c id p r e c ip ita b le
c o u n ts p e r 2 - m l. in c u b a tio n , s e e F ig . 10.
U pon te rm in a tio n o f th e 3 7 °C in c u b a tio n , th e e n tire 2 -m l. sa m p le
w a s lo a d e d in to 2 - m l. B eckm an c e ll u lo s e n itr a te c e n trifu g e tu b e s (S to ck
# 3 0 3 3 6 9 ). T h e se s m a ll c e n tr ifu g e tu b e s w e re m a in ta in e d a t s a l t / i c e
b a th te m p e ra tu re s th ro u g h o u t th e lo a d in g p r o c e d u r e .
The c a p s w e re tig h te n e d , th e tu b e s p la c e d in th e T i-5 0 ro to r
a d a p t o r s , a n d th e s a m p le c e n trifu g e d fo r 2 h o u rs a t 40,000 RPM (105,000
xg) in th e B eckm an m o d e l L ty p e T i-5 0 ro to r.
The s u p e r n a ta n t from th is c e n tr ifu g a tio n w as c a r e f u lly rem o v ed
w ith a P a s te u r p ip e tte a n d tr a n s f e r r e d to a c le a n 12- m l. c o n ic a l c e n tr i­
fu g e t u b e . The p e l l e t w a s th e n g en tly , r in s e d w ith b u ffe r LMSN a n d th e
w ash s a v e d .
The p e l l e t w a s f in a lly d is s o lv e d in 1.0 m l. o f g la s s d i s ­
t i l l e d w a te r a n d s e t a s i d e a t 2 °C fo r l a t e r u s e .
T hree m illig ra m s o f ly s o z y m e w e re a d d e d to th e 105,000 xg s u p e r ­
n a ta n t (I mg ./ m l . d i s t i l l e d w a ter) a s a p ro te in c a r r ie r . A te n - f o ld e x c e ss of
12
C - a r g in in e an d ly s in e w a s a d d e d (0.1 m l. o f a s o lu tio n o f
7.31 m g. ly s in e a n d 8.25 m g. a rg in in e to e a c h 1.0 m l. o f s u p e r n a ta n t) . The
m ix tu re w a s s tir r e d a n d s u b s e q u e n tly p r e c ip ita te d by a d d in g 1.49 m l. o f
48
100% s a tu r a te d am m onium s u lf a te to e a c h m l. o f s o lu tio n (60% fin a l
s a tu r a tio n ) . The c lo u d y w h ite s u s p e n s io n w a s a llo w e d to f lo c c u la te a t
2 °C fo r 30 m in u te s .
The p r e c ip ita te w a s c o lle c te d b y c e n tr ifu g a tio n a t to p s p e e d in a
c lin ic a l c e n trifu g e a t 2 °C for 2 0 m in u te s . The s u p e r n a ta n t w a s d i s ­
c a rd e d a n d th e p r e c ip ita te w a s h e d tw ic e w ith 60% s a tu r a te d am m onium
s u lf a te by r e s u s p e n s io n a n d r e c e n tr if u g a tio n . The p r e c ip ita te w as
f in a lly s u s p e n d e d in 1.0 m l. o f g la s s d i s t i l l e d w a t e r .
S ix m illilite r s o f 5% tr ic h lo r o a c e tic a c id w a s n e x t a d d e d an d th e
s o lu tio n h e a te d a t 9 0 °C for 15 m in u te s to h y d ro ly z e th e e s te r if ie d b o n d s
o f th e t-RN A m o ie tie s . A fter c o o lin g a t 2 °C fo r 20 m in u te s , th e p r e c ip i­
t a t e w a s a g a in c o lle c te d by c e n tr if u g a tio n . D is c a rd in g th is s u p e r n a ta n t,
th e p r e c ip ita te w a s w a s h e d tw ic e a t room te m p e ra tu re w ith 5% tr ic h lo r o ­
a c e t i c a c id by r e s u s p e n s io n a n d r e c e n tr if u g a tio n .
The r e s u ltin g p r e c i p i t a t e , s t i l l m o is t from th e 5% tr ic h lo r o a c e tic
a c id w a s h , w a s w a s h e d tw ic e w ith 6.0 m l. o f an e q u a l- p a r ts s o lu tio n o f
re a g e n t g ra d e e t h a n o l / e t h e r , fo llo w e d b y a fin a l w a s h o f 6.0 m l. o f r e ­
a g e n t g ra d e e th e r . T his f in a l w a s h e d p r e c ip ita te w a s c o lle c te d by c e n ­
tr ifu g a tio n a n d th e s u p e r n a ta n t w a s d is c a r d e d .
The w a s h e d p ro d u c t w a s d rie d u n d e r a j e t of n itro g e n a t 2 °C and
s to re d a t - 9 0 ° C in th e ReVco c o ld c h e s t u n til u s e d .
The p r e c ip ita te w h ic h r e s u lte d from th e i n itia l 105,000 xg c e n tr if u ­
g a tio n ( d is s o lv e d in th e 1.0 m l. o f d i s t i l l e d w a te r), w a s tr e a te d in an
49
id e n tic a l m a n n er a s th e s u p e r n a ta n t. T h is w a s h e d a n d d rie d p ro d u c t w a s
a l s o s to re d a t - 9 O0 C u n til u s e d .
P a rt 2: O x id a tio n an d tr y p tic d ig e s tio n o f MS2 c o a t p ro te in —
n a tiv e a n d in v i t r o .
The fo llo w in g p ro c e d u re in v o lv e d th e u s e o f e ith e r
p u rifie d n a tiv e MS2 c o a t p ro te in o r th e w a s h e d a n d d rie d M S2 ^RNA in
v itr o p e p t i d e s . In ,the c a s e o f n a tiv e MS2 c o a t p r o t e i n , th e p u re p ro d u c t
a lo n e w a s u s e d , w h e re a s w ith a n in v itro p ro d u c t a d d itio n a l n a tiv e MS2
c o a t p ro te in (or ly so z y m e ) h a d b e e n a d d e d a s a c a r r i e r .
Two m illilite r s o f r e a g e n t g ra d e fo rm ic a c id an d 0.1 m l. o f 30%
r e a g e n t g ra d e h y d ro g e n p e ro x id e w e re a d d e d to a 12- m l. c o n ic a l c e n tr i­
fu g e t u b e . T he m ix tu re w a s a llo w e d to s i t in a w a te ry ic e b a th fo r tw o
h o u rs a t w h ic h tim e th e t i t e r o f p e ro x y fo rm ic a c id w a s a t a m axim um .
T h r e e - te n th s m illilite r o f th is liq u id w a s a d d e d to 3 m g. o f th e
d rie d p r o te in to b e o x id iz e d a n d a llo w e d to s i t in th e ic e b a th for o n e
h o u r. The r e a c tio n w a s s to p p e d by th e a d d itio n o f 1.0 m l. o f c h ille d d i s ­
t i l l e d w a te r an d th e m ix tu re im m e d ia te ly Iy o p h y H z e d to d r y n e s s .
O ne m illilite r o f 0.05 M am m onium b ic a r b o n a te a n d 0.12 m g. o f
TPCK tr e a te d try p s in w e re a d d e d to th e d rie d o x id iz e d p ro d u c t. The
m ix tu re w a s in c u b a te d a t room te m p e ra tu re fo r th re e h o u rs , fo llo w e d b y
ly o p h y liz a tio n to d ry n e s s in a 2 5 0 -m l. te a rd ro p ly o p h y liz a tio n f la s k .
C a re w a s ta k e n a t th is ju n c tu re to p re v e n t th e e x tre m e ly lig h t
ly o p h y liz e d p o w d e r from b e in g d raw n in to th e ly o p h y liz a tio n a p p a r a t u s .
T h is w a s a c c o m p lis h e d by b lo c k in g th e n e c k o f th e te a rd ro p fla s k w ith
50
a fin e T eflo n s c r e e n an d in a ll c a s e s a v o id in g th e u s e o f c o n ic a l c e n ­
trifu g e tu b e s for ly o p h y liz a tio n . R e p e a te d th a w in g a n d fre e z in g o f th e
ly o p h y liz a tio n s a m p le d u rin g th is p r o c e s s to a llo w th e r e s id u e to b e ­
com e m ore c o n c e n tr a te d a l s o a p p e a re d to h e lp in t h i s r e g a r d . The p ro ­
d u c t w a s s to re d a t - 9 0 ° C in th e R evco c o ld c h e s t u n til fin g e rp rin tin g
w a s p e rfo rm e d .
/
P a rt III: F in g e rp rin tin g p ro c e d u re .
I.
E le c tr o p h o r e s is .
T w enty to 40 |il. o f g la s s d i s t i l l e d w a te r w as
a d d e d to th e d r ie d , o x id iz e d a n d d ig e s te d p ro d u c t a n d th e sa m p le d i s ­
s o lv e d so th a t th e r e s u ltin g s o lu tio n w a s w ith o u t a n y p r e c ip ita te . In
som e c a s e s th e p ro d u c t w o u ld n o t d is s o lv e c o m p le te ly a n d a p p ro x i­
m a te ly I p i. o f 1% N H ^O H w a s a d d e d to f a c i l i t a t e s o lu b ility . In som e
in s t a n c e s th e d rie d p ro d u c t w a s d is s o lv e d d ir e c tly in b u ffe r E B N -I. The
r e s u l t s , h o w e v e r, w e re id e n tic a l no m a tte r w h ic h m eth o d w a s e m p lo y e d .
U sin g a 5 -|u l. m ic r o p ip e tte , th e sa m p le w as c a r e f u lly a p p lie d to
th e corner, o f a 30 cm . x 40 cm . p ie c e o f W h atm an 3 -m m . f il te r p a p e r ,
ta k in g c a re th a t th e fo llo w in g c r ite r ia w e re m et:
1 . The sa m p le a p p lie d w a s a lw a y s w ith o u t a n y v is ib le p r e c ip i­
ta t e .
2 . The fin a l s p o t w a s a lw a y s b e tw e e n 0.4 cm . an d 0.6 cm . in
d ia m e te r .
3 . F ive m ic ro lite rs w a s a p p lie d fo llo w e d by a ir d ry in g for 15 m in ­
u te s b e fo re a p p ly in g th e n e x t 5 p i.
, 51
4 . The f i l t e r p a p e r w a s n o t to rn d u rin g th e p r o c e s s o f s p o ttin g .
5 . The f il te r p a p e r w a s a lw a y s p la c e d on a s h e e t o f c le a n p la s t ic
d u rin g s p o ttin g .
6. The s a m p le w a s a p p lie d to th e o rig in a s a 5 -|j,l. drop an d n o t
a s a c o n tin u o u s flo w .
A fter th e a p p lie d sa m p le h a d b e e n a llo w e d to a ir d ry fo r a t l e a s t
I h o u r, th e f il te r p.aper w a s la id o n to a la rg e p ie c e o f c le a n c le a r p l a s ­
tic .
The p a p e r w a s now c o m p le te ly m o is te n e d w ith b u ffe r EBN-I u sin g a
1 0 -m l. p ip e tte . The sa m p le s p o t i t s e l f w a s m o is te n e d b y c a r e fu lly
a llo w in g b u ffe r to d iff u s e a lo n g fo u r d ir e c tio n s to w a rd s th e c e n te r of
th e s p o t. The f ilte r p a p e r w a s th e n im m e d ia te ly b lo tte d b y p la c in g i t on
a la r g e r s h e e t of f il te r p a p e r (W hatm an N o . 3mm) an d c o v e rin g i t w ith a
s e c o n d la rg e s h e e t o f th e sa m e f i l t e r p a p e r . A 6" w id e r o lle r (M a y fie ld
C o ., I n c .) w a s th e n ro lle d o v e r th e f il te r p a p e r , an d th e s a n d w ic h e d
f il te r p a p e r th o ro u g h ly b lo tte d . A s e c o n d to p an d b o tto m s h e e t of
W h atm an N o . 3mm f il te r p a p e r w a s e x c h a n g e d fo r th e n o w w e t s h e e ts
a n d b lo ttin g re su m e d for a b o u t o n e m in u te .
The b lo tte d f il te r p a p e r w a s n e x t p o s itio n e d on th e b ottom o f a
w a te r c o o le d e le c tr o p h o r e s is p la te an d th e c e ll u lo s e s e p a r a to r s and
W h atm an #1 w ic k s m o u n ted (s e e F ig . 7 ). B ufferE B N -I w a s a d d e d to th e
e le c tr o p h o r e s is b u ffe r ta n k s an d a llo w e d to s e e k th e sa m e le v e l by
v irtu e o f a tu b e jo in in g b o th t a n k s . A s h e e t o f p l a s t i c w a s th e n p la c e d
o v e r th e m o is te n e d an d b lo tte d f il te r p a p e r an d th e to p p la te w a s
52
lo w e re d a n d c la m p e d in to p l a c e . The s y s te m w as th e n a llo w e d to
e q u ilib r a te fo r o n e - h a l f h o u r. The tu b e jo in in g th e tw o b u ffe r ta n k s w a s
th e n p in c h e d o ff w ith a s c r e w c la m p .
E le c tr o p h o r e s is c o n tin u e d fo r 2.5 h o u rs a t a v o lta g e flu x o f 30 v/cm .
(1100 v o lts to ta l) , 25 m illia m p e re s c u r r e n t. The u n it w a s d is a rm e d o f th e
h ig h v o lta g e an d th e e le c tro p h o re to g ra m re m o v e d . The p a p e r w a s f ir s t
d rie d a t room te m p e ra tu re fo r 15 m in u te s fo llo w e d b y d ry in g in an o v en
a t 7 0 °C fo r 30 m in u te s .
2.
C h ro m a to g ra p h y w a s th e n p e rfo rm e d .
C h ro m a to g ra p h y . A c y lin d e r w a s form ed o u t o f th e d rie d
e le c tro p h o re to g ra m b y c o n n e c tin g p o in ts A-A a n d B-B w ith p l a s t i c p a p e r
c lip s ( s e e F ig . 7 ). The tu b e w a s th e n p la c e d (e d g e A-A dow n) in to a
ro u n d g l a s s c h ro m a to g ra p h y t n a k , 3 0 c m . in d ia m e te r a n d 60 cm . in
h e ig h t'. In th e b o tto m o f th e ta n k w a s p la c e d a 1 0 -c m . g l a s s P e tri d is h
c o n ta in in g a b o u t 50 m l. o f b u ffe r C B N -I. A g la s s c o v e r w a s p la c e d o v e r
th e ta n k a n d v a p o r e q u ilib r a tio n w a s a llo w e d to p ro c e e d fo r 12 h o u r s .
At th e te rm in a tio n o f e q u ilib r a tio n , 2 00 m l. o f b u ffe r C BN -I w as
in tr o d u c e d in to th e b o tto m o f th e ta n k w ith a 4 0 -c m . lo n g stem m ed fu n '
n e l . A sc e n d in g c h ro m a to g ra p h y w a s a llo w e d to p ro g r e s s fo r 8 to 10
h o u r s . The ch ro m ato g ram w a s th e n re m o v e d an d d rie d a s w a s th e
e le c tr o p h o r e to g r a m .
The d rie d fin g e rp rin t w a s q u ic k ly d ip p e d in to a 0.25% s o lu tio n o f
n in h y d rin in a c e t o n e , a n d th e n d rie d a t 7 0 °C for 10 m in u te s . The r e s u l t ­
in g n in h y d rin s p o ts (ly s o z y m e or MS2 c o a t p ro te in ) g a v e a q u ic k c h e c k
I
53
a s to th e s e p a r a tio n o f th e tr y p tic s p o t s . V is u a liz a tio n o f th e
in
v itro p e p tid e s w a s n e x t a c h ie v e d b y a u to ra d io g ra p h y .
3 . A u to ra d io g ra p h y . A s h e e t o f 8" x lO " Kodak X -ra y film w as
p la c e d n e x t to th e s u r f a c e o f th e d ry f in g e r p r in t, th e s h e e ts p la c e d in to
a n a p p ro p ria te X -ra y film h o ld e r a n d th e film a llo w e d to e x p o s e for 10 to
15 d a y s .
The film w a s d e v e lo p e d u s in g Kodak R apid X -ra y d e v e lo p e r an d
th e s iz e a n d d is tr ib u tio n o f th e e x p o s e d s p o ts w a s n o te d b o th on th e
X -ra y film an d th e fin g e rp rin t i t s e l f .
4 . S c in tilla tio n c o u n tin g o f
14
C - tr y p tic p e p tid e s . The in d iv id u a l
r a d io a c tiv e s p o ts w e re c u t o u t o f th e f il te r p a p e r f in g e r p r in t and th e
ra d io a c tiv e le v e l d e te rm in e d by o n e o f tw o c o u n tin g p r o c e d u r e s . E ith e r
m e th o d g a v e p ro p o r tio n a l- r e s u lts w h en co m p ared to th e o th e r m e th o d .
M eth o d A. The
14
C ra d io a c tiv e f il te r p a p e r d i s c s w e re p la c e d in to
n e w 2 5 - m l. B eckm an s c in t illa tio n v ia ls a n d 2 0 m l. o f s c i n t illa tio n flu id
a d d e d (42 m l. S p e c tra flu o r PPO -PO PO P to 958 m l. o f t o l u l e n e ) . R ad io ­
a c tiv e l e v e l s w e re th e n r e g is te r e d on th e N u c le a r - C h ic a g o m odel 6801
liq u id s c i n t i l l a t i o n c o u n te r .
M eth o d B. The r a d io a c tiv e m a te ria l w a s e lu te d from e a c h d is c
w ith 20 m l. o f g l a s s d i s t i l l e d w a te r a n d th e e lu e n t p la c e d in to Beckm an
2 5 -m l. s c i n t illa tio n v i a l s .
The a q u e o u s, s o lu tio n w a s th e n d rie d a t
7 0 °C an d th e r e s id u e s u b s e q u e n tly s o lu b iliz e d by th e a d d itio n o f I m l.
/
o f H y d ro x id e o f H y a m in e . The s c i n t i l l a t i o n flu id w a s a d d e d (20 m b.) an d
r a d io a c tiv e c o u n tin g re c o rd e d a s in M eth o d A.
54
In c o rp o ra tio n w ith p u rifie d rib o s o m e s an d in itia tio n fa c to rs
In v itro in c o rp o ra tio n s u s in g p u rifie d rib o s o m e s , in itia tio n f a c ­
to rs an d MS2 m e s s e n g e r RNA w e re c a r rie d o u t in 0 .5 -m l. o r 1 .0 -m l.
i
v o lu m e s . The c o n c e n tr a tio n s o f th e v a rio u s r e a g e n ts a d d e d w e re a s
f o llo w s . All r e a g e n ts w e re m ade up in b u ffe r L M SN -16.
0.025 m l. o f 0.08 M ATP
0.025 m l. o f 0.005 M PEP
.
0.02 5 m l. o f 5 XlO- 4 M GTP/CTP
0.02 5 m l. o f 30.00 IX J/m l. p y ru v a te k in a s e
0.050 m l. o f 10 [iC i/m l. ^4C - I y s in e
,
14
0.050 m l. TO |J.C i/m l. C - a rg in in e
_3
0.025 m l. o f a s o lu tio n 10 M in e a c h L -am in o a c id e x c e p t h i s t i ­
d in e , a rg in in e a n d ly s in e w h ic h w e re d e le te d ■
0.025 m l. o f 30 m g ./m l. w a s h e d rib o s o m e s
0.050 ml,. o f 10 A2 qq u n i t s / m l . c ru d e i n i tia tio n f a c to rs
0.050 m l. o f b u ffe r LM SN-1 6
0.050 m l. o f I m g ./m l. MS2 RNA
0.050 m l. o f 4 m g ./m l. E . p o ll tRNA
0.050 m l. o f pH 5 fra c tio n
' E ach o n e - m illi lite r in c o rp o ra tio n th e re fo re c o n ta in e d th e fo llo w ­
in g i n g r e d i e n t s .
4.0 HM ATP
0.02 5 [iM GTP/CTP ■
'
I
J*: «•••'
55
PEP
0.25
5x10
“3
|_iM e a c h L -a m ln o a c i d ( e x c e p t h i s t i d i n e , a r g in in e a n d
ly s in e )
1.5 IU p y r u v a te k i n a s e
1.0 HCi
14
C -Iy sin e
1.0 HCi ^ C - a r g i n i n e
400 HQ. E. c o li t-RNA
100 HO. MS2 RNA
0.5 Ag go u n its c ru d e i n i t i a t i o n fa c to r s
750 HO- w a s h e d rib o s o m e s
I Ag go u n its pH 5 fr a c tio n
10 HM Tris
80 HM N H 4 C l
.
16.0 HM Mg(OAc) 2
' E a c h i n c u b a t io n tu b e w a s s u p p l i e d w ith a ll i n g r e d i e n t s e x c e p t th e
MS2 J11RNA. ' S u f f ic ie n t b u ffe r Was th e n a d d e d to b rin g th e t o t a l volum e
o f e a c h i n c u b a t i o n m ix tu re to 1.0 m l. W h en a ll th e t u b e s w e re r e a d y ,
MS2 J11RNA w a s a d d e d u s in g a r i b o n u c l e a s e free lOO-Hl. E p p en d o rf
m i c r o p i p e t t e . The m ix tu re w a s t h e n 'g e n t l y a g i t a t e d u t i l i z i n g a V ortex Jr.
m ix e r a n d th e tu b e w a s p l a c e d in a 3 7 o c w a t e r b a th fo r th e a p p r o p r ia te
le n g th o f tim e .
O ne h u n d re d fif ty m ic ro g ra m s o f b o v in e serum a lb u m in w a s a d d e d
a s a p r o te in c a r r i e r (o p tio n a l) p rio r to p r e c i p i t a t i o n o f t h e r a d io a c ti v e
56
p r o d u c ts w ith .th e a d d itio n o f 6.0 m l. o f 5% t r i c h l o r o a c e t i c a c i d (for a
LO-ml. incorporation).
The m ix tu re w a s a g i t a t e d w ith a V ortex Jr. m i x e r '
a n d h e a te d to 9 0 ° C for 15 m i n u t e s . • A fter c o o lin g a t 2 0 c fo r 30 m i n u t e s ,
t h e p r e c i p i t a t e w a s f il te r e d on M illip o re HAWP 2.5 cm . n i t r o c e l l u l o s e
f i l t e r s . The f i l t e r s w e re n e x t d rie d a t 7 0 ° C for 15 m i n u t e s . R ad io ­
a c t i v i t y w a s d e te r m in e d by c o u n tin g on a N u c l e a r - C h i c a g o m o d el 4312
3 - c m . g a s flo w c o u n te r .
T y p ic a l i n c u b a t i o n s g a v e o v e r 2500 cp m /0 .5 m l. u s in g 100 [ug. o f
MS2 mRNA. B ackground r a d i o a c t i v i t y a v e r a g e d 50 to 100 c p m . A g r e a t e r
in c o r p o r a tio n w a s s o m e tim e s d e s i r e d a n d o b ta in e d b y th e a d d itio n o f
la r g e r a m o u n ts o f MS2 mRNA.
■■
'T
-------
4.
EXPERIMENTAL RESULTS
(a) In v itro fin g e r p r in ts
D e v e lo p m e n t o f th e s y s te m
E ffe c t o f Mg+ + c o n c e n tr a tio n on S -3 0 i n c o r p o r a t i o n s .
The ra te of
fo rm a tio n o f a c i d p r e c i p i t a b l e r a d i o a c t i v i t y v a r i e s w h e n th e Mg++ c o n ­
c e n t r a t i o n i s v a r ie d in a n in v itro p r o te in s y n t h e s i z i n g s y s t e m .
N a t h a n s , N o t a n i , S c h w a rz & Z in d er (1962) h a v e fo u n d t h a t I l m M Mg++
c o n c e n t r a t i o n r e s u l t e d in m a x im a l in c o r p o r a tio n o f ^ C - a m i n o a c i d s in
an S -3 0 a s s i s t e d MS2 mRNA d i r e c t e d s y s t e m .
C la rk (19 65) f o u n d , h o w ­
e v e r , t h a t 8 mM Mg++ w a s optim um u n d e r th e s a m e c o n d i t i o n s . The
e f f e c t s sh o w n in F ig . 8 w ere p ro d u c e d w h e n t h e m a g n e s iu m io n c o n c e n ­
tr a t i o n o f a t y p i c a l S -3 0 i n c u b a t io n w a s v a r ie d o v e r th e ra n g e o f 7 mM
to 16 m M . The v a l u e o f 8 mM to 9 mM m a g n e s iu m w a s th u s c o n s id e r e d
optim um for a n MS2 d i r e c t e d S -3 0 i n c o r p o r a t i o n , a n d a l l r e a g e n t s w e re
m a d e up in a b u ffe r c o n ta i n in g 8.5 mM m a g n e s iu m (b u ffer L M S N ).
K in e tic s o f
14
C -a m in o a c i d i n c o r p o r a t i o n .
It c a n be e s t i m a t e d
t h a t th e tim e r e q u ir e d for th e c o m p le tio n o f a n MS2 c o a t p r o te in m o le ­
c u le i s a b o u t 3 to 5 s e c o n d s a t 3 7 ° C ( G o l d s t e i n , 19 64). In v itro s t u d i e s
in d e e d i n d i c a t e t h a t th e r e i s an i n c r e a s e in t h e a c id p r e c i p i t a b l e r a d i o ­
a c t i v i t y a s a fu n c tio n o f ti m e , b u t th e r e l a t i o n s h i p i s n o t l i n e a r .
In a n e ffo rt to d e te r m in e th e k i n e t i c s o f an MS2 mRNA d i r e c te d
S -3 0 a s s i s t e d s y s t e m , s a m p l e s w e re s i m u l t a n e o u s l y s t a r t e d b y th e
,j:
58
a d d itio n o f MS2 mRNA a n d s to p p e d a t s p e c i f i e d tim e s b y th e a d d itio n o f
5% t r i c h l o r o a c e t i c a c i d .
The r e s u l t s a re sh o w n in F ig . 9 .
The m o s t
e f f i c i e n t in c u b a t io n p e r io d w a s 3 0 to 40 m inutes-, a s th e ^ C - a m i n o a c i d
in c o r p o r a tio n p l a t e a u e d a f t e r t h i s tim e . A c c o r d i n g ly , a l l e n s u i n g i n c o r ­
p o r a tio n s w e re in c u b a t e d for 40 m i n u t e s .
S tim u la to ry e f f e c t o f MS2 m e s s e n g e r RNA.
V arying a m o u n ts o f
th e RNA w e re a d d e d to a n S -3 0 s y s te m in a n a tte m p t to m e a s u r e th e
s t im u la to r y e f f e c t o f p h a g e MS2 mRNA d i r e c t e d in cop o ra tio n o f ^ C am in o a c i d s . As c a n be s e e n from F ig . 10, a c l e a r s t i m u l a t i o n w a s p ro ­
d u c e d a t 2 0 ia g ./m l. F u rth e rm o re , th e m e s s a g e - d e p e n d e n t MS2 mRNA
d ire c ted
14
C - a m in o a c i d in c o r p o r a tio n w a s i n c r e a s e d more th a n te n fo ld
o v e r a 20 p g . / m l . c o n tro l by th e a d d itio n o f 200 p g . / m l . o f mRMA. From
t h i s e x p e r im e n t , 100 |j.g./:ml. w a s c o n s i d e r e d a c o n c e n t r a t i o n o f m e s s a g e
h ig h e n o u g h to p ro d u c e s t a t i s t i c a l l y a c c u r a t e c o u n t s , y e t w a s e c o n o m i­
c a l in t h e u s e o f MS2 mRNA.
D e p e n d e n c e o f in c o r p o r a tio n on a d d e d
14
C -a m in o a c i d s . In o rd e r
to b e s t g a u g e th e m o s t e f f i c i e n t c o n c e n t r a t i o n o f ^ C - a r g i n i n e a n d ^ C l y s i n e p r e s e n t in a n i n c u b a t i o n , th e c o n c e n tr a tio n o f t h e s e ^ C - a m i n o
a c i d s w a s - v a r i e d from 0.1 p C i / m l . to 1.0 p .C i/m l. As c a n b e s e e n in F ig .
1 1 , t h e a m o u n t o f a c i d p r e c i p i t a b l e m a te r ia l i n c r e a s e d a s th e c o n c e n t r a ­
tio n o f a d d e d r a d i o a c t i v e am ino a c i d s i n c r e a s e d . It w a s c o n c lu d e d t h a t
I
59
to m in im iz e th e lim itin g n a tu re o f th e a d d e d ^ C - a m i n o a c i d s , 1.0 (J.Ci o f
l y s i n e a n d a r g in in e w o u ld b e a d d e d to e a c h 1.0- m l . i n c u b a t i o n .
E ffe c t o f c h lo r a m p h e n ic o l on t o t a l a c i d p r e c i p i t a b l e c o u n t s . The
p e r c e n t in h ib iti o n o f t o t a l a c i d p r e c i p i t a b l e c o u n ts w h e n c h lo r a m p h e n i­
c o l (CAP) i s a d d e d to a p r o te in s y n t h e s i z i n g s y s te m v a r i e s w ith th e
p o ly n u c le o tid e b e in g u s e d a s a m e s s a g e . J u lia n (19 65) s h o w e d th a t
67 [jig./ml. o f CAP w a s s u f f i c i e n t to p ro d u c e 60% i n h ib iti o n o f p ro te in
s y n t h e s i s in a p o l y a d e n y l i c a c i d d i r e c t e d s y s t e m , w h e r e a s H ahn &
W o lfe (19 65) d e m o n s t r a t e d a re q u ir e m e n t o f 300 p g . / m l . o f CAP to g iv e
80% i n h ib iti o n in a p o ly u r i d y lic a c i d d i r e c t e d s y s t e m .
To d e te r m in e CAP'S e f f e c t on th e S- 3 0 a s s i s t e d s y s t e m , v a ry in g
a m o u n ts o f CAP w e re a d d e d to s t a n d a r d in c u b a t io n t u b e s .
From th e
g ra p h sh o w n in F ig . 12 i t i s e v i d e n t t h a t a c o n c e n tr a tio n o f o n ly
2.5 p g ./m l. o f c h lo r a m p h e n ic o l p r o d u c e d a 50% i n h ib iti o n o f t o t a l a c id
p re c ip ita b le c o u n ts .
E ffe c t o f c h lo r a m p h e n ic o l on p e p t i d e s r e l e a s e d in v i t r o .
MS2
s p e c i f i c p o ly p e p tid e c h a i n s tKat h a v e b e e n c o m p le te d a n d r e l e a s e d from
t h e rib o s o m a l c o m p le x a re te rm e d " r e l e a s e d " p e p t i d e s .
T h o se p e p t i d e s
re m a in in g a t t a c h e d to th e rib o s o m a l c o m p le x a re te rm e d "b o u n d "
p e p tid e s.
An e x p e r im e n t w a s d e s i g n e d to e x a m in e th e e f f e c t o f CAP on r e ­
l e a s e d p e p t i d e s . A 2 .0 -m l. s a m p le w a s i n c u b a t e d , w ith a l i q u o t s r e ­
m oved a t v a ry in g t i m e s , c e n tr if u g e d a t 105,000 xg for 2 h o u rs a n d th e
sV ■
:....
60
s u p e r n a t a n t e x a m in e d for a c i d p r e c i p i t a b l e c o u n t s .
F ig . 13 d e p i c t s th e
r e s u l t s o f s u c h a n e x p e r im e n t . From t h i s o n e m ay c o n c lu d e th a t th e
a p p e a r a n c e o f r e l e a s e d p e p t i d e s w a s i n h i b i t e d by c h lo r a m p h e n ic o l.
P e p tid e map o f n a t i v e MS2 c o a t p r o t e i n . A t r y p t i c fin g e rp r in t w a s
m ade o f 3.0 m g. o f n a t i v e MS2 c o a t p r o te in to d e te r m in e th e n u m b er a n d
d i s t r i b u t i o n o f tr y p t ic p e p t i d e s .
The d e t a i l s o f o x i d a t i o n , d i g e s t i o n a n d
fin g e r p r in tin g h a v e b e e n p r e v i o u s l y d e s c r i b e d in " M a t e r i a l s a n d M e th ­
o d s " . The m e th o d o lo g y fo llo w e d t h a t o f N a th a n s (19 65), a lth o u g h th e
14
C - I a b e l e d am in o a c i d s w e re d if f e r e n t.
The r e s u l t i n g fin g e r p r in t i s d e ­
p i c t e d in F ig . 14, a s a t r a c i n g o f th e o r i g i n a l . f i n g e r p r i n t . E lev e n n i n h y d rin p o s i t i v e s p o t s w e re p r o d u c e d .
P o s itio n 5 w a s a t t r i b u t e d to free
l y s i n e , a s c o n firm e d b y a n in d e p e n d e n t fin g e rp r in t o f l y s i n e a l o n e .
P e p tid e map o f .in v itr o MS2 d i r e c t e d r e l e a s e d p e p t i d e s . F in g e r­
p r in tin g w a s p e rfo rm ed on th e 105,000x g s u p e r n a t a n t o f a 2 .0 -m l. i n c u ­
b a tio n to c o m p a re th e n u m b e r a n d d i s t r i b u t i o n o f tr y p t i c p e p t i d e s 'i n th e
r e l e a s e d in v itr o c o a t p r o t e i n . Three m g. o f ly s o z y m e w e re a d d e d a s a
p r o te in c a r r i e r a n d a s t a n d a r d p e p tid e m ap w a s m a d e . A fter e x p o s u r e to
X -ra y film for o n e w e e k , a t r a c in g w a s m ad e o f th e r a d i o - o p a q u e a r e a s .
F ig . 15 d e p i c t s t h i s p e p tid e m a p . A gain th e n u m b e r a n d d is t r i b u t i o n of
s p o t s c l o s e l y a p p r o x im a te s th
,e o f th e n a tiv e c o a t p r o t e i n . The n u m ­
b e rin g s e q u e n c e o f th e s p o t s i s a n a lo g o u s to th e n a t i v e c o a t p ro te in
p ep tid e m a p .
61
E ffe c t o f c h lo r a m p h e n ic o l on th e p e p tid e map o f p e p t i d e s r e l e a s e d
in v i t r o .
The r e l e a s e d in v itr o p e p t i d e s a re in h ib ite d b y CAP a t a c o n ­
c e n t r a t i o n o f 2.5 n g ./m l., a s sh o w n in F ig . 13. F i g s . 17 a n d 19 sh o w t h a t
t h i s i n h ib iti o n i s a l s o a p p l i e d to th e rib o s o m a l b o u n d m a te r ia l a n d th a t
a l l rib o s o m a l b o u n d tr y p t ic p e p t i d e s a re e q u a l l y i n h i b i t e d .
A fin g e r p r in t w a s m ad e o f th e 105,000 x g s u p e r n a t a n t o f a 2 .0 -m l.
. in c u b a t i o n to d e te r m in e th e e f f e c t o f CAP on th e r e l e a s e d in v itro p e p ­
tid e s.
The r e s u l t s , d e p i c t e d in F ig . 16, a g a in s h o w th e s a m e n u m ber
a n d d i s t r i b u t i o n o f s p o t s . A q u a n t i t a t i v e a n a l y s i s o f e a c h s p o t by
s c i n t i l l a t i o n c o u n tin g y ie ld e d r e s u l t s q u a l i t a t i v e l y p r o p o r tio n a l w ith
t h o s e o f F ig . 2 0 . As w ith th e rib o s o m a l b o u n d p e p t i d e s , r e l e a s e d t r y p ­
t i c p e p t i d e s w e re i n h i b i t e d p r o p o r tio n a l ly .
T h e s e r e s u l t s confirm th o s e
o f N a th a n s (19 65).
E x p e rim e n ta l r e s u l t s
E ffe c t o f c h lo r a m p h e n ic o l on a c i d p r e c i p i t a b l e c o u n ts o f th e
r ib o s o m a l b o u n d in v itr o p e p t i d e s .
This e x p e r im e n t w a s d e s i g n e d to x
e x a m in e th e e f f e c t o f c h lo r a m p h e n c io l on th e fo rm atio n o f MS2 mRNA
s p e c i f i c r ib o s o m a l b o u n d p o l y p e p tid e c h a i n s . The p r e c e d u r e w a s
i d e n t i c a l to t h a t o f F ig . 13.
F ig . 17 d e p i c t s th e r e s u l t s o f t h i s e x p e r im e n t , i n d i c a t i n g a l o w - .
' e r e d q u a n t i t y o f r a d i o a c t i v i t y a s s o c i a t e d w ith th e r ib o s o m a l b o u n d
p e p t i d e s in th e p r e s e n c e o f CAP. T his s u g g e s t e d t h a t th e l e v e l o f
-
62
C - n a s c e n t p e p t i d e s a s s o c i a t e d w ith th e rib o s o m e w a s r e ­
duced.
P e p tid e map o f th e in v itro MS2 d i r e c t e d rib o s o m a l b o u n d
p e p t i d e s . A t r y p t i c fin g e rp r in t w a s m a d e o f th e 105,000 x g in v itro
]4
C-
l a b e l e d p r o d u c t to d e te r m in e t h e n a tu r e o f th e in v itro p r o d u c ts p ro ­
d u c e d u n d e r th e d i r e c t i o n o f MS2 mRNA. The in c u b a t io n w a s a s t a n d a r d
2.0- m l . s a m p le in c u b a t e d a n d p r e p a r e d a s d e s c r i b e d in " M a t e r i a l s a n d
M e th o d s " . The s u p e r n a t a n t from th e 105,000 x g c e n tr if u g a tio n w a s s a v e d
fo r l a t e r u s e .
F ig . 18 s h o w s a t r a c i n g o f a fin g e r p r in t a u to r a d io g r a p h o f th e in
v itr o i n c o r p o r a t i o n . The n u m b e r a n d d i s t r i b u t i o n o f t h e s e s p o t s c l o s e l y
a p p r o a c h th e fin g e r p r in t o f th e n a t i v e MS2 c o a t p r o t e i n . O n e a d d itio n a l
\
s p o t c a n b e i d e n t i f i e d a s b e lo n g in g to fre e a r g i n i n e , a re m n a n t o f th e
14
C - a m in o a c i d s a d d e d d u rin g th e i n c u b a t i o n . It c a n b e c o n c lu d e d from
t h i s fin g e r p r in t t h a t th e m a jo r p r o d u c t p ro d u c e d in v itr o w a s MS2 c o a t
p ro te in .
E ffe c t o f c h lo r a m p h e n ic o l o n th e p e p tid e map o f th e rib o s o m a l
b o u n d in v itro p e p t i d e s . At a c o n c e n t r a t i o n o f 2.5 p g . / m l . , CAP in h i b i t s
50% o f th e t o t a l a c i d p r e c i p i t a b l e
14
C - a m in o a c i d l a b e l e d m a te r ia l (F ig .
12). A s a m p le w a s in c u b a t e d w ith 2.5 jag./ml. o f c h lo r a m p h e n ic o l to d e ­
te rm in e CAP'S e f f e c t on t h e in v itro p e p t i d e s . A p e p t i d e map w a s m ad e
63
o f th e 105,000 x g p r e c i p i t a t e a n d an a u to r a d io g r a p h p ro d u c e d b y e x p o s ­
in g Kodak X -ra y film to th e f in g e r p r in t.
F ig . 19 i s a t r a c i n g o f th e r a d i o - o p a q u e a r e a s o f th e a u t o r a d i o ­
g ra m . It, i s q u ite a p p a r e n t from t h i s t h a t th e n u m b e r a n d d i s t r i b u t i o n o f
th e t r y p t i c p e p t i d e s re m a in th e s a m e a s . t h e fin g e rp r in t o f n a t i v e c o a t
p r o t e i n . Each r a d i o a c t i v e s p o t w a s c o u n te d to co m p are th e d if f e r e n c e s
in r a d i o a c t i v i t y b e tw e e n th e c o n tro l in v itr o tr y p t i c p e p t i d e s a n d th o s e
in v itr o tr y p t ic p e p t i d e s p ro d u c e d in th e p r e s e n c e o f 2.5 p g ./m l. CAP'. As
e x p l a i n e d in d e t a i l in " M a t e r i a l s , a n d M e th o d s " , th e a r e a s on th e f in g e r ­
p r in t c o r r e s p o n d i n g to r a d i o - o p a q u e a r e a s on th e X -ra y film w e re c u t
o u t a n d th e r a d i o a c t i v i t y d e te r m in e d by liq u id s c i n t i l l a t i o n c o u n tin g .
The r e s u l t s a re s u m m a riz e d in F ig . 2 0 .
The r a d i o a c t i v i t y in e a c h s p o t
w a s r e d u c e d by a p r o p o r tio n a l ly e q u a l a m o u n t. All tr y p t ic p e p t i d e s w e re
i n h i b i t e d e q u a l l y , p ro d u c in g no c h a n g e in th e C to N te r m in a l p e p tid e
g r a d ie n t o f l a b e l i n g . T h e s e r e s u l t s s u g g e s t GAP's e f f e c t w a s on p e p ­
tid e c h a in i n i t i a t i o n a n d n o t p e p tid e c h a i n e x t e n s i o n . (b) The ro le o f rib o s o m a l w a s h f a c t o r s
Mg++ t i t r a t i o n c u rv e o f th e p u r if ie d in v itro s y s t e m . S e v e ra l
g ro u p s h a v e r e p o r te d t h a t th e Mg+ + c o n c e n tr a tio n optim um s h i f t s to a
h ig h e r v a lu e w h e n p u r if ie d rib o s o m e s a re u s e d in c o n ju n c t io n w ith
i n i t i a t i o n f a c to r s in a n in v itr o n a t u r a l mRNA s t im u la te d s y s t e m a s c o m ­
p a r e d to a c ru d e 8 - 3 0 a s s i s t e d s y s t e m (R e v e l, b e lo n g , Braw erm an &
*’'*
='
--
.
>
— - ' •; '--I-.-,..
‘,
- • .u ..
— : ■-
64
G ros , 19 68; R e v e l , H e rz b e rg , B e c a re v ic & G ros , 19 68; W a h b a , C h ae ,
I w a s a k l , M a z u m d e r & M i l l e r e t a l . , 1969; K o la k o f s k y , 1 9 6 6 ). In lig h t
o f t h i s p o s s i b i l i t y , in c u b a t i o n s o f p u r if ie d r i b o s o m e s , h ig h s p e e d , ,
:
s u p e r n a t a n t f a c to r s in th e form o f a pH 5' fr a c tio n ( J u lia n , 1965), a n d
++
MS2 mRNA w e re c a r r ie d o u t a t v a ry in g c o n c e n t r a t i o n s o f Mg
to d e t e r ­
m ine th e optim um c o n c e n t r a t i o n o f Mg++. F ig . 21 d e p i c t s th e r e s u l t s o f
t h i s e x p e r im e n t , a n d s h o w s an o p tim a l r a d i o a c t i v e in c o r p o r a tio n a t
++
16 mM Mg
z
.
. A c c o rd in g ly , a ll fu rth e r in c u b a t i o n s w e re c a r r ie d o u t a t
,
16 mM Mg+"1". At t h i s c o n c e n t r a t i o n o f Mg++, th e s y s te m i s e n t i r e l y d e ­
p e n d e n t u p o n a d d e d i n i t i a t i o n fa c to r s (F ig . 22).
H e n c e f o r th , a n y r e f e r e n c e to a p u r if ie d s y s te m r e f e r s to an in
v itro s y s t e m c o n ta i n in g DEAE w a s h e d r i b o s o m e s , a d d e d c ru d e i n i t i a t i o n
f a c t o r s , a n energy, s o u r c e , p u r if ie d MS2 mRNA a n d a pH 5' f r a c tio n .
T itra tio n c u rv e o f c ru d e i n i t i a t i o n fa c to r s a g a i n s t th e p u rif ie d
sy ste m .
The a d d itio n o f i n c r e a s i n g a m o u n ts o f MS2 mRNA to an in v itr o
s y s te m p r o d u c e s a p ro p o r tio n a l i n c r e a s e in th e in c o r p o r a tio n o f
am in o a c i d s .
14
C-
S u ch a r e s u l t p ro b a b ly i n d i c a t e s th e lim it in g n a tu re o f th e
mRNA. S in c e a s i m i l a r r e l a t i o n s h i p m ig h t e x i s t w ith r e s p e c t to c ru d e
|!
i n i t i a t i o n f a c to r s , a t i t r a t i o n c u rv e o f c ru d e i n i t i a t i o n f a c t o s w a s p e r ­
form ed to d e te r m in e th e optimum c o n c e n tr a tio n o f t h e s e f a c t o r s . The
c ru d e f a c to r s e x h i b i t e d a n Aggp/^ZBO r a t i o ° f 1:2 w h e n p r e p a r e d a c c o r d ­
in g to th e d e t a i l s g iv e n in " M a t e r i a l s a n d M e th o d s " .
i
’ .
.
> - C H C
'i
"W
if! -I—
. I
—
65
F ig . 22 d e p i c t s t h i s t i t r a t i o n c u r v e . T h ro u g h o u t th e ra n g e of c o n ­
c e n t r a t i o n s u s e d , th e a d d itio n o f c ru d e i n i t i a t i o n f a c to r s c a u s e d a p r o I
p o r ti o n a l i n c r e a s e in t o t a l a c i d p r e c p i t a b l e c o u n t s . A c c o rd in g ly , in a ll
fu rth e r i n c o r p o r a t i o n s , 0.5 AggQ u n its o f i n i t i a t i o n f a c to r s w e re u s e d p e r
m l. o f in c o r p o r a tio n s a m p l e .
K in e tic s o f
I
14
C - a m in o a c id in c o r p o r a tio n o f th e p u r if ie d s y s t e m .
In F ig . 9 , th e k i n e t i c s o f ^ C - a m i n o a c i d in c o r p o r a tio n o f t h e S -3 0 s y s tern w a s i n v e s t i g a t e d . O v e r 80% o f th e
14
C -a m in o a c i d in c o r p o r a tin g
a b i l i t y re m a in e d in s u c h a s y s te m a f t e r th e f i r s t te n m in u te s o f i n c u b a ­
tio n .
S u ch r e s i d u a l in c o r p o r a tin g a b i l i t y r e p r e s e n t s b o th e lo n g a t io n a n d
r e - i n i t i a t i o n c o l l e c t i v e l y . It w a s d e c i d e d to d e te r m in e th e r e s i d u a l
^ C - a m i n o a c i d i n c o r p o r a tin g p o t e n t i a l o f t h e p u rif ie d s y s t e m .
F ig . 23 d e p i c t s th e k i n e t i c s o f ^ C - a m i n o a c id in c o r p o r a t i o n . All
s a m p l e s w e re t r e a t e d i d e n t i c a l l y a n d th g in c u b a t io n s te r m in a te d a t th e
i n d i c a t e d tim e s b y th e a d d itio n o f 5% t r i c h l o r o a c e t i c a c i d . I t i s c l e a r
from t h i s g ra p h t h a t o n ly 20 % o f th e r a d i o a c t i v i t y w a s in c o r p o r a te d in
t h e f i r s t fiv e m i n u t e s .
E ffe c t o f c h lo r a m p h e n ic o l on t o t a l a c i d p r e c i p i t a b l e c o u n ts o f th e
p u r if ie d s y s t e m . An S - 3 0 a s s i s t e d MS2 mRNA d i r e c t e d in v itr o s y s te m
i s i n h i b i t e d 50% a t a c o n c e n tr a tio n o f c h lo ra m p h e n ic o l e q u a l to
2.5 (ig./m l. (F ig . 12). V arying c o n c e n t r a t i o n s o f CAP w e re th e r e f o r e t e s t ­
ed w ith th e p u rif ie d s y s te m to m ak e a c o m p a r is o n w ith th e S -3 0 s y s t e m .
V ;-
V -
.
.
V.
‘" - r - r - ------ .-S —
- • .-.V
66
F ig . 24 d e p i c t s t h i s e x p e r im e n t .
C h lo ra m p h e n ic o l w a s more th a n tw ic e
Y
a s e f f e c t i v e in th e p u r if ie d s y s te m a s in th e S -3 0 s y s t e m , re q u irin g
o n ly 0.9 p g . / m l . CAP c o n c e n t r a t i o n to y ie ld a 50% i n h i b i t i o n .
E ffe c t o f c ru d e i n i t i a t i o n f a c to r s on a p u rif ie d s y s t e m r e - i n c u b a t e d
a f t e r a tw o - h o u r c e n t r i f u g a t i o n .
The p r e s e n c e o f c ru d e i n i t i a t i o n f a c ­
to r s in a p u r if ie d s y s t e m i s a n e c e s s a r y a d j u n c t for th e s u c c e s s f u l i n ­
c o rp o ra tio n o f ^ C - a m i n o a c i d s . W h en s u c h f a c to r s a re w i t h h e l d ,
n e g l i g i b l e a m o u n ts o f a m in o a c i d s a re in c o r p o r a te d . I t w a s d e c id e d to
i n v e s t i g a t e th e ro le o f c ru d e i n i t i a t i o n fa c to r s in a p u r if ie d s y s t e m .
One m i l l i l i t e r of a s ta n d a r d in c u b a t i o n m ixture
w a s i n c u b a t e d for
5 m in u te s a t 3 7 ° C to i n i t i a t e th e t r a n s l a t i o n o f mRNA. S u b s e q u e n t l y ,
e a c h m ix tu re w a s q u ic k ly c h i l l e d to' O0 C in a s a l t / i c e b a t h a n d c e n t r i ­
fu g e d a t 105,000 xg for 2 h o u rs in a 2 .0 -m l. Beckm an c e n tr if u g e tu b e c o n ­
t a i n i n g a 0.2- m l . p a d o f 10% s u c r o s e . Thus th e s o lu b le i n i t i a t i o n f a c ­
to r s w e re re m o v e d from th e r ib o s o m a l a g g r e g a t e . U pon c o m p le tio n o f
th e c e n t r i f u g a t i o n , th e 10% s u c r o s e p a d p lu s 0.1 m l. o f s u p e r n a t a n t
d i r e c t l y a b o v e th e p a d w a s t r a n s f e r r e d to a m ix tu re c o n ta i n in g a ll i n c u ­
b a tio n r e a g e n t s e x c e p t r ib o s o m e s a n d m e s s a g e .
The s y s t e m r e a d i l y i n ­
c o r p o r a te d a d d i t i o n a l ^ C - a m i n o a c i d s w h en t r a n s f e r r e d to a m ixture
c o n ta i n in g c ru d e i n i t i a t i o n f a c to r s (F ig . 2 5 ). There w a s a 65% l o s s o f
in c o r p o r a tin g a b i l i t y i f th e r e - i n c u b a t i o n w a s l a c k i n g i n i t i a t i o n f a c t o r s .
This s u b s t a n t i a l l o s s in in c o r p o r a tin g a b i l i t y w a s th e r e f o r e a r e s u l t o f
th e a b s e n c e o f c ru d e i n i t i a t i o n f a c t o r s .
67
It m ay be c o n c lu d e d t h a t t h i s in c o r p o r a tin g s y s te m c a n be r e ­
i n i t i a t e d from p o ly s d m a l c o m p le x e s t h a t w e re w a s h e d fre e o f in i t i a t i o n
f a c t o r s . Such r e - i n i t i a t i o n i s d e p e n d e n t upon a d d itio n o f c ru d e i n i ­
t i a t i o n f a c to r s n o t found in th e pH 5' p r e c i p i t a t e .
The la c k o f c ru d e
'
i n i t i a t i o n f a c to r s in th e 105,000 xg p e l l e t c l e a r l y r e s u l t s in th e l o s s o f
^ C - a m i n o a c i d in c o r p o r a t i o n . T his s y s t e m a llo w s th e d if f e r e n t i a t i o n of
o f e lo n g a t io n a n d i n i t i a t i o n . The 65% g a in o f r a d i o a c t i v i t y in F ig . 25 i s
th u s e n t i r e l y d u e to i n i t i a t i o n c a t a l y z e d b y th e p r e s e n c e o f c ru d e i n i tia tio n f a c to r s .
D e p e n d e n c e o f th e p u r if ie d s y s te m on th e pH 5' f r a c t i o n . A lthough
th e c ru d e i n i t i a t i o n f a c to r p r e p a r a t io n p r o b a b ly d iff e r s from t h o s e e n -
'f
;
z y m e s found in t h e pH 5' f r a c t i o n , t h e p o s s i b i l i t y o f c o m p le m e n ta ry
e n z y m e s c a n n o t be ru le d o u t . The r e - i n i t i a t e d rib o s o m a l p e l l e t w o u ld
n o t b e d e p e n d e n t s o l e l y on i n i t i a t i o n f a c to r s i f t h i s w e re t r u e .
In F ig . 2 6 , 1 .0 -m l. s a m p l e s w e re i n c u b a t e d for 5 m in u te s to b e g in
i n i t i a t i o n , c e n tr if u g e d a t 105,000 x g to p e l l e t th e r ib o s o m a l b o u n d p e p ­
t i d e s a n d t h e rib o s o m a l m a te r ia l r e - i n c u b a t e d in a s t a n d a r d m ix tu re
m in u s r ib o s o m e s a n d m e s s e n g e r . The s y s t e m w a s c o m p le te a n d fu rth e r
in c o r p o r a tio n o c c u r r e d w h e n b o th i n i t i a t i o n fa c to rs a n d pH 5' p r e c i p i ­
t a t e w e re p r e s e n t . W h e n , h o w e v e r , th e pH 5' p r e c i p i t a t e w a s o m itte d ,
,!
1
th e s y s t e m w o u ld n o t c o n tin u e a m in o a c i d i n c o r p o r a t i o n . Thus th e tw o
e n z y m e p r e p a r a t i o n s a re c o m p le m e n ta ry . The i n i t i a t i o n f a c to r p r e p a r a ­
t io n a lo n e w ill n o t s u p p o r t p ro te in s y n t h e s i s in a p u r if ie d s y s te m ..
'I
/
y
------------------- - 7 —
;
f
■■
T .
.
T - h
- -
68
E ffe c t o f c h lo ra m p h e n ic o l on i n i t i a t i o n f a c to r d e p e n d e n t a n d i n d e pendent
14
C -a m in o a c i d i n c o r p o r a t i o n . I t i s n o w f e a s i b l e to t e s t GAP's
a c t i o n on o u r s y s t e m w h ic h i s d e p e n d e n t upon a d d e d r ib o s o m a l w a s h
f a c t o r s . F ig . 2 7 r e p r e s e n t s t h i s e x p e r im e n t.
In t h i s e x p e r i m e n t , p u rif ie d in c u b a t io n m ix tu r e s c o n ta i n in g a ll
i n g r e d i e n t s e x c e p t ^ C - a r g i n i n e a n d ^4C - I y s i n e (b u t in c lu d in g rib o s o m a l
w a s h fa c to r s ) w e re i n c u b a t e d for fiv e m in u te s a t 3 7 ° C .
The 1 .0-m l.
m ix tu r e s w e re th e n lo a d e d in to 2.0—m l. B eckm an c e l l u l o s e n i t r a t e c e n t r i ­
fuge t u b e s ' c o n ta i n in g a 0.2 - m l . p a d o f 10% s u c r o s e ( s p e c i a l s u c r o s e
d e n s i t y g rad e) in b u ffe r L M S N . The m a te r ia l w a s c e n tr if u g e d for tw o
h o u rs a t 105,000 x g in th e M o d e l L-2 u l t r a c e n t r i f u g e . The p ro c e d u re
th u s fa r w a s a n a lo g o u s to F ig . 2 5 . U pon c o m p le tio n o f this, c e n t r i f u g a ­
t i o n , 0.3 m l . o f th e s u c r o s e p a d w a s tr a n s f e r r e d to a p u r if ie d in c u b a t io n
s y s te m w ith
14
14
C - a r g i n i n e a n d C - I y s i n e p r e s e n t a n d re - i n c u b a t e d for
2 5 m in u te s a t 3 7 ° C .
• In o n e c a s e , th e r e - in c u b a te ,d m ix tu re c o n ta i n e d a c o m p le te c o m ­
p le m e n t o f r e a g e n t s in c lu d in g r ib o s o m a l w a s h f a c t o r s . As e x p e c t e d , i t
in c o r p o r a te d *4C - a m in o a c i d s a n d p r o d u c e d 1710 CPM (F ig . 2 7). W hen
1.0 pig./m l. o f CAP w a s a d d e d to a r e - i n c u b a t i o n m ix tu re c o n ta i n in g r i b o - '
s o m a l w a s h f a c t o r s , th e in c o r p o r a tio n w a s r e d u c e d to 763 CPM (F ig . 2 7),
r e p r e s e n t i n g a 45% i n h i b i t i o n .
S u ch r e s u l t s w o u ld b e e x p e c t e d i f e i t h e r i n i t i a t i o n o r e lo n g a tio n
w e re i n h i b i t e d . The r e - i n c u b a t e d s y s t e m a llo w e d b o th i n i t i a t i o n and
...
. — y ’;
.L -
r
69
e lo n g a t io n to p r o c e e d .
CAP m ig h t, h o w e v e r , h a v e a c t e d a t e i t h e r s t e p .
I n i t i a t i o n a n d e lo n g a t io n c a n be d i f f e r e n t i a t e d by i n v e s t i g a t i n g GAP's .
e f f e c t on th e r e s i d u a l
14
C -a m in o a c i d in c o r p o r a tin g p o t e n t i a l w h en r e ­
i n c u b a t io n o c c u r s in th e a b s e n c e o f r ib o s o m a l w a s h f a c t o r s . Such
-V. .
r e s i d u a l in c o r p o r a tin g p o t e n t i a l m ay be a tt r i b u t e d to p e p tid e c h a in
e x ten sio n .
' F ig . 2 7 a l s o r e p r e s e n t s an e x p e r im e n t in w h ic h r ib o s o m a l c o m i
p l e x e s w e re r e - i n c u b a t e d w ith o u t rib o s o m a l w a s h f a c t o r s p r e s e n t ,
14
p ro d u c in g 398 C P M . S in c e C - a m in o a c i d s w e re a d d e d o n ly d u rin g th e
2 5 - m in u te r e i n c u b a t i o n , t h e s e c o u n ts r e p r e s e n t p e p tid e c h a in e x t e n s i o n
a l o n e , a s th e s y s te m w a s w ith o u t c ru d e i n i t i a t i o n f a c t o r s . W hen
1.0 p g . / m l . o f CAP w a s a d d e d to th e r e - i n c u b a t i o n no i n h ib iti o n w a s
n o te d .
Ten |_ig./ml. o f CAP a g a in p ro d u c e d a n e g lib le i n h ib iti o n d ro p ­
p in g th e in c o r p o r a tio n to 379 CPM (F ig . 27). Such a c o n c e n tr a tio n o f
CAP w o u ld b e e x p e c t e d to p ro d u c e a 100% in h ib iti o n o f t h e s y s t e m .
T h e s e r e s u l t s s u g g e s t t h a t CAP h a s a s i g n i f i c a n t l y s tr o n g e r e f f e c t on
i n i t i a t i o n a s c o m p a re d to e lo n g a t i o n .
5.
DISCUSSION
The p r e c e d in g e x p e r im e n ts i n d i c a t e t h a t a t lo w c o n c e n t r a t i o n s o i
c h lo r a m p h e n ic o l (CAP), i n h ib iti o n o f p r o te in s y n t h e s i s i s a t th e l e v e l o f
i n i t i a t i o n in a n a tu r a l mRNA d i r e c t e d s y s t e m .
F u r th e r m o re , s u c h i n h i b i -
tio n i s d i r e c t e d a t i n i t i a t i o n f a c to r d e p e n d e n t in c o r p o r a tio n o f
14
C -a m in o
a c id s.
J u lia n (19 65) s h o w e d t h a t fo rm atio n o f th e d i - a n d t r i - l y s i n e p e p ­
t i d e s w a s n o t a f f e c t e d b y CAP, w h e r e a s fo rm atio n o f th e la r g e r p e p t i d e s
w a s in h ib ite d .. He s u g g e s t e d t h a t CAP a c t e d a t a p e p t i d e b o n d forming
ste p .
T raut & M unro (19 64) found t h a t CAP in h ib ite d t h e e f f e c t o f puro^-
m y c in in r e l e a s i n g p h e n y l a l a n i n e p e p t i d e s from p o ly p h e n y la la n y l- s R N A
a t t a c h e d to r i b o s o m e s . On th e b a s i s o f t h i s fin d in g th e y s u g g e s t e d t h a t
CAP a c t e d a s a n i n h i b i t o r o f th e p e p t i d e b o n d form ing e n z y m e , a t th e
s a m e s i t e a s p u ro m y c in . W e i s b e r g e r (1967) h a s s u g g e s t e d t h a t CAP
a c t e d on a r ib o s o m a l s i t e a t a s t a g e a f t e r b in d in g o f mRNA a n d d u rin g
p e p tid e s y n t h e s i s to p r e v e n t th e f in a l c o n d e n s a t i o n o f a m in o a c i d s a n d
t h e g ro w th o f p o ly p e p tid e c h a i n s . Thus th e g e n e r a l c o n s e n s u s w a s t h a t
CAP a c t e d o n th e e lo n g a t io n p r o c e s s in p r o te in s y n t h e s i s .
A rm entrout & W e i s b e r g e r (19 67) h a d p r e v io u s ly c o n c lu d e d t h a t
p r o te in s y n t h e s i s d i r e c t e d by p o ly u r i d y lic a c i d w a s m ore r e s i s t a n t to
CAP i n h ib iti o n th an t h a t in d u c e d by n a tu r a l ^RNA, a n d s u g g e s t e d t h a t
th e in h ib ito r y e f f e c t o f CAP m ay be p e c u l i a r to th e RNA p r e p a r a t io n
-CL*.-;
--
v
4 ,"V. *>' T-”
71
used.
S in c e th e w ork p r e v i o u s l y d e s c r i b e d u t i l i z e d s y n t h e t i c mRNAs, i t
w a s n o w d e s i r a b l e to t e s t th is m o d e l o f e lo n g a t io n i n h ib iti o n on a n in
v itro s y s te m d i r e c t e d b y a n a tu r a l mRNA.
The t r a n s l a t i o n o f th e c o a t p r o te in c i s t r o n o f th e MS2 v iru s
.
n a t u r a l mRNA m ay b e e n v i s i o n e d a s in v o lv in g a p o p u la tio n o f r i b o s o m e /
m e s s e n g e r c o m p le x e s in a l l s t a g e s of t r a n s l a t i o n .
C o n se q u e n tly , every
c o m p le x w ill h a v e in c o r p o r a te d a n N - t e r m i n a l am ino a c i d , b u t o n ly
t h o s e c o m p le x e s in th e fin a l s t a g e s o f p o ly p e p tid e s y n t h e s i s w ill h a v e
in c o r p o r a te d a C - t e r m i n a l a m in o a c i d . If CAP w e re to blo ck , a s te p in
e l o n g a t i o n , w e w o u ld e x p e c t a p o p u la tio n o f r i b o s o m e / m e s s e n g e r c o m ­
p l e x e s fro z e n in t h e v a r io u s s t a g e s o f e l o n g a t i o n . H e n c e , w e m ig h t n o t
14
e x p e c t a n y d e c r e a s e in th e rib o s o m a l C - n a s c e n t p e p t i d e s a s c o m p a re d
to a n o rm a l p r o te in s y n t h e s i z i n g s y s t e m . F u r th e r m o r e , a s e lo n g a t io n
w a s i n h i b i t e d on a mRNA s t r a n d , a n i n c r e a s e in t h e q u a n t i t y o f th e N te r m in a l p e p t i d e s m ig h t b e e x p e c t e d a s n e w ly i n i t i a t e d r ib o s o m e s w e re
a llo w e d to s y n t h e s i z e p e p t i d e s b e fo re b e in g i n h i b i t e d in so m e s t a g e o f
elo n g atio n .
N a t h a n s (1965) e x a m in e d t h e e f f e c t o f CAP on th e r e l e a s e d R e ­
l a b e l e d MS2 in v itr o p e p t i d e s . H is m e th o d o lo g y u t i l i z e d a fin g e rp r in t o f
a t r y p t i c d i g e s t o f th e n o n - r ib o s o m a l b o u n d
p e p tid e s.
14
C - I a b e l e d MS2 in v itro
He fo u n d no i n c r e a s e in th e tr y p t ic N - t e r m i n a l p e p t i d e s o v e r
th e C - t e r m i n a l p e p t i d e s w h e n s u c h p e p t i d e s w e re s y n t h e s i z e d in th e
p r e s e n c e o f SiS n g ./m l. o f CAP. T his w a s in c o n t r a s t to th e r e l e a s e d
72
■ p ep tid es form ed in .the p r e s e n c e o f p u r o m y c in , w h ic h s h o w e d a n i n - . •
c r e a s e in th e n u m b e r o f N - te r m in a l p e p t i d e s .
T h e s e r e s u l t s w e re in
a c c o r d w ith a n e lo n g a t io n i n h ib iti o n m o d el for CAP, a s o n e w o u ld e x ­
p e c t th e r e l e a s e d p e p t i d e s to be w h o le a n d p o s s e s s an e q u a l n u m b er o f
N a n d C - t e f m i n a l p e p t i d e s . H is w ork a l s o te n d e d to ru le o u t CAP i n ­
d u c e d p re m a tu re r e l e a s e , for in t h i s c a s e w e s h o u ld e x p e c t r e s u l t s
s im i l a r to t h o s e s e e n w ith th e r e l e a s e d p e p t i d e s s y n t h e s i z e d in th e
p r e s e n c e o f 'p u ro m y c in . H o w e v e r, he d id n o t e x a m in e CAP'S e f f e c t on
th e r ib o s o m a l b o u n d
14
C -n a sc e n t p e p tid e s.
The tru e t e s t for a n e l o n g a ­
tio n i n h ib iti o n m o d el e x i s t s h e r e , s i n c e th e p e p t i d e s r e s u l t i n g from
i n h ib iti o n w o u ld l i k e l y b e tr a p p e d on th e m e s s e n g e r / r i b o s o m e c o m p le x .
It w a s d e c i d e d to c o m p a re th e r ib o s o m a l b o u n d to r e l e a s e d p ro d u c ts
u s in g N a t h a n 's p r o c e d u r e s .
A n o rm al d i s t r i b u t i o n o f tr y p t i c p e p t i d e s r e s u l t e d w h e n a p e p tid e
map w a s m ade o f t r y p t i c h y d r o l y s a t e s o f r e l e a s e d p e p t i d e s s y n t h e s i z e d
in th e p r e s e n c e o f 2.5 p g ./m l. o f CAP, w ith no p r e p o n d e r a n c e o f a n y
tr y p t ic p e p tid e o v e r a n y o th e r t r y p t ic p e p t i d e (F ig . 16). T h e s e r e s u l t s
confirm t h o s e p r e s e n t e d b y N a th a n s (1965) on th e r e l e a s e d MS2 in v itr o
p e p tid e s.
Thuszw e m ay ru le o u t CAP i n d u c e d p re m a tu re r e l e a s e , b u t
o ffe r no c o n c l u s i o n s r e g a rd in g a n e lo n g a t io n in h ib iti o n m o d e l for th e
m e c h a n is m o f a c t i o n in n a tu r a l mRNA d i r e c t e d s y s te m s .
A p e p t i d e map w a s m ade o f th e 105>000 xg ^ C - I y s i n e a n d ^ C a r g in in e l a b e l e d p r o d u c ts a s s o c i a t e d w ith th e p r e c i p i t a t e o f a n in v itro
,-r-
73
MS2 d i r e c t e d s y s t e m . S u ch p r o d u c ts w e re a llo w e d to b e s y n t h e s i z e d in
th e p r e s e n c e o f 2.5 p g ./m l. o f CAP. A d i s t r i b u t i o n o f t r y p t i c p e p tid e s
i d e n t i c a l to t h a t o f n a t i v e MS2 c o a t p r o te in w a s p r o d u c e d . F i g . '19. As
t h i s m a te r ia l p r im a rily r e p r e s e n t s n a s c e n t p e p t i d e s ( N a t h a n s , N o t a n i ,
S c h w a rz & Z i n d e r , 19 62; N a t h a n s , 19 65), th e r e s u l t s a r e in a c c o r d w ith
a m o d el o f n o rm al g ro w in g p e p tid e . c h a i n s . A g a in , t h e n o rm a l d i s t r i b u ­
tio n a n d n u m b e r o f t r y p t i c p e p t i d e s r u l e s o u t p re m a tu re r e l e a s e of
n a sc e n t p e p tid e s.
A q u a n t i t a t i v e a n a l y s i s o f th e r a d i o a c t i v i t y o f e a c h t r y p t i c p e p tid e
r e v e a l e d t h a t th e r e w a s a p r o p o r tio n a l d e c r e a s e in t h e r a d i o a c t i v i t y o f
a l l th e tr y p t i c p e p t i d e s a n d th e r e w a s no c h a n g e in th e q u a n tity o f N te r m in a l p e p t i d e s in th e p r e s e n c e o f CAP, F ig . 2 0 . T o ta l a c i d p r e c i p i t a b l e c o u n ts a re i n h i b i t e d b y 50% a t a c o n c e n t r a t i o n o f CAP o f 2.5 [ig./ml.
S e v e n t y - f i v e p e r c e n t o f t h i s i n h ib iti o n o c c u r s in th e r ib o s o m a l b o u n d
m a t e r i a l , w h e r e a s th e r e l e a s e d m a te r ia l i s i n h i b i t e d by o n ly 25%.
T h e re fo re th e p rim ary e f f e c t o f CAP o n th e MS2 mRNA d i r e c t e d s y s te m is
t h e r e d u c tio n o f r ib o s o m a l b o u n d ^ C - I a b e l e d m a t e r i a l .
T his i s c l e a r l y
n o t in a g r e e m e n t w ith a m o d e l o f e lo n g a t io n i n h i b i t i o n .
' A c o n f l i c t th u s e x i s t s b e tw e e n th e r e s u l t s o f th is s tu d y a n d t h a t
m o d e l g e n e r a l l y a s c r i b e d to CAP'S a c t i o n . To e x a m in e t h e s e c o n f l i c t s ,
i t i s h e lp fu l to c o n s i d e r o th e r m e c h a n is m s t h a t m ay a c c o u n t for th e i n ­
h ib itio n o f p r o te in s y n t h e s i s b y CAP in th e MS2 mRNA d i r e c t e d s y s t e m .
W e m ay ru le o u t i n h ib iti o n o f t e r m i n a t i o n , a s th e r e l e a s e o f
14
C -Ia b e led
74
m a te r ia l i s f a c i l i t a t e d r a t h e r t h a n i n h i b i t e d . W e h a v e p r e v i o u s l y d i s ­
c o u n te d p re m a tu re r e l e a s e . S in c e th e d a ta p r e s e n t e d in t h i s s tu d y do
n o t s u p p o r t a n e lo n g a t io n i n h ib iti o n m o d e l, we a re fo rc e d to fin a lly
c o n sid e r in itia tio n .
If CAP w e re to b lo c k a s t e p in i n i t i a t i o n , p r e v i o u s l y i n i t i a t e d
r i b o s o m e / m e s s e n g e r c o m p le x e s w o u ld b e a llo w e d to c o m p le te t r a n s l a tio n of th e mRNA. Any
14
C - I a b e l e d n a s c e n t p e p t i d e s w o u ld b e r e l e a s e d
a n d l e a v e t h e mRNA in e i t h e r a n i n h i b i t e d s t a t e o f i n i t i a t i o n or p r e v e n t
a n y i n i t i a t i o n c o m p le x e s from form ing a t a l l . The n e t e f f e c t w o u ld b e to
14
d e c r e a s e t h e r ib o s o m a l b o u n d C - I a b e l e d n a s c e n t p e p t i d e s . W e w o u ld
■j,
;
n o t e x p e c t a n y i n c r e a s e in th e n u m b e r o f N - te r m i n a l n a s c e n t p e p tid e s
o v e r a c o n t r o l , a s a n y n a s c e n t m a te r ia l re m a in in g w o u ld b e in a n o rm al
p r o c e s s o f p e p t i d e c h a in e l o n g a t i o n . T h e r e f o r e , in p a r t i a l l y in h ib ito r y
c o n c e n t r a t i o n s o f CAP, a p e r c e n t a g e o f th e r i b o s o m e / m e s s e n g e r c o m 1
14
p l e x e s w o u ld b e s tr ip p e d o f C - I a b e l e d m a t e r i a l , a n d t h e re m a in in g
u n i n h ib ite d f r a c tio n w o u ld a p p e a r a s n o rm a lly g ro w in g n a s c e n t m a t e r i a l .
The d a t a p r e s e n t e d in t h i s s tu d y s tr o n g ly s u p p o r t t h i s m o d e l o f i n h i b i ­
tio n o f p r o te in s y n t h e s i s b y CAP.
A p p ro x im a te ly o n e y e a r a f t e r t h i s e f f e c t on i n i t i a t i o n o f th e MS2
s y s t e m w a s d i s c o v e r e d , Irv in & J u lia n (1970) p u b l i s h e d t h e i r fin d in g s
on th e e f f e c t o f CAP on th e p o l y c y t i d y l i c a c id d i r e c t e d s y n t h e s i s o f
14
C - p o l y p r o l i n e . They n o te d t h a t n o t o n ly w a s t h i s s y s t e m s i g n i f i c a n t l y
m ore s e n s i t i v e to CAP t h a n o th e r s y n t h e t i c mRNAs, b u t t h a t CAP
--ry
75
i n h i b i t e d th e s y n t h e s i s o f a l l p e p t i d e s in c lu d in g th e d i - a n d t r i - p r o l i n e
r e s i d u e s . This w a s i n . c o n t r a s t to th e p o l y a d e n y l i c a c i d a n d p o ly u r id y lic
a c i d d i r e c t e d s y s t e m s w h e re CAP i n h i b i t e d o n ly p e p t i d e s la r g e r th a n th e
x t r i - p e p t i d e ( J u l i a n , 1965; P e s t k a , 19 6 9 ). Irv in a n d J u lia n c o n c lu d e d
t h a t t h i s e f f e c t w a s a t t r i b u t a b l e to i n h ib iti o n o f a n i n i t i a t i o n s te p a n d
not
p e p tid e c h a in e x t e n s i o n a s i t a p p e a r s t o . a c t in th e p o ly a d e n y l ic
(
a c i d a n d p o ly u r i d y lic a c i d d i r e c t e d s y s t e m s .
The CAP s e n s i t i v i t y o f th e MS2 mRNA a n d p o l y c y t i d y l i c a c id
d i r e c t e d s y s t e m s s u g g e s t t h a t th e m ode o f a c ti o n o f th e drug d iffe rs
. from th e in h ib ito r y m e c h a n is m o b s e r v e d in th e l e s s s e n s i t i v e s y n t h e t i c
JtlRNA d i r e c t e d s y s t e m s . It i s g e n e r a l l y a c c e p t e d t h a t . i n h i b i t i o n i s a t
t h e l e v e l o f e l o n g a t io n in th e p o l y a d e n y l i c a c i d a n d p o ly u r i d y lic a c id
d i r e c t e d s y s t e m s , w h e n h ig h l e v e l s o f CAP a re u s e d . In th e MS2 mRNA
a n d p o ly c y tid y lic a c i d d i r e c t e d s y s t e m s , t h e e v i d e n c e p r e s e n t e d in t h i s
d i s c u s s i o n s u g g e s t s i n h ib iti o n o f a n i n i t i a t i o n s t e p .
It i s illu m i n a ti n g to c o n s i d e r th e l e v e l s o f CAP r e q u ir e d to i n h i b i t
t h e p o l y a d e n y l i c a c i d a n d p o ly u r i d y lic a c i d d i r e c t e d . s y s t e m s v e r s u s th e
n a t u r a l mRNA a n d p o ly c y tid y lic a c i d d i r e c t e d s y s t e m s .
This e v id e n c e
p o in ts to tw o p o s s i b l e m e c h a n is m s o f i n h ib iti o n ( e lo n g a tio n a n d i n i ­
t i a t i o n ) . The l e v e l s o f CAP r e q u ir e d .for in h ib iti o n fa ll in to tw o g ro u p s
a n d fu r th e r s u g g e s t th a t, o n e m e c h a n is m o f in h ib iti o n i s a lo w c o n c e n tr a ­
tio n p h e n o m e n o n w h ile th e s e c o n d e f f e c t i s found o n ly a t m u ch h ig h e r
c o n c e n t r a t i o n s o f th e d ru g . In a d d i t i o n , s y s t e m s w h ic h r e q u ire
76
rib o s o m e w a s h f a c to r s a p p e a r to b e m o s t s e n s i t i v e to th e d ru g . At a
c o n c e n t r a t i o n o f CAP of 2.5 |ag./m l. t o t a l MS2
C -p e p tid e s y n th e s is is
i n h i b i t e d 50% w h e n a s s i s t e d by a n -S-SO e x t r a c t . S im ila r ly , o n ly
2.5 (ig ./m l. o f CAP a re re q u ir e d to b rin g a b o u t 60% i n h ib iti o n o f th e p o l y c y t i d y l i c a c i d d i r e c t e d s y s te m (Irvin & J u l i a n , 1 9 7 0 ). O n t h e o th e r
h a n d , 67 |ag./m l. o f CAP a re r e q u ir e d to i n h i b i t b y 60% the- p o ly a d e n y l ic
a c i d d i r e c t e d s y s t e m ( J u l i a n , 1 9 6 5 ). At a c o n c e n tr a tio n o f CAP of
180 Mg./m l . , t o t a l p h e n y l a l a n i n e p e p t i d e s y n t h e s i s i s u n in h ib ite d
( P e s t k a , 1 9 6 9 ). The d i s t i n c t e f f e c t s a t th e tw o l e v e l s s u g g e s t t h a t a t
lo w c o n c e n t r a t i o n s o f CAP i n i t i a t i o n i s i n h i b i t e d , a s in th e MS2 mRNA
a n d p o l y c y t i d y l i c a c i d d i r e c t e d s y s t e m s , a n d a t h ig h c o n c e n t r a t i o n s
e lo n g a t io n i s e f f e c t e d a s w ith th e o th e r tw o s y n t h e t i c mRNAs. This
s u g g e s t s t h a t th e r e a re s i m i l a r m e c h a n is m s o f i n i t i a t i o n in th e MS2 a n d
p o l y c y t i d y l i c a c i d d i r e c t e d s y s t e m s w h ic h a re d if f e r e n t from th e o th e r
tw o s y n t h e t i c mR NAs.
In 1 9 7 1 , C a m e ro n , R ogers ,& J u lia n n o te d t h a t t h e a d d itio n of
c ru d e r ib o s o m a l w a s h f a c to r s s t im u la te d t h e in c o r p o r a tio n o f ^ C p ro lin e in a p o l y c y t i d y l i c a c i d d i r e c t e d s y s t e m . In a d d i t i o n , a c ru d e
h ig h s a l t r ib o s o m a l w a s h f r a c tio n w a s r e q u ir e d a s a s u p p le m e n t w h e n a
pH 5' f r a c tio n w a s u s e d a s a s o u r c e o f s u p e r n a t a n t f a c t o r s in th e p o l y ­
c y t i d y l i c a c i d d i r e c t e d s y s t e m . In c o n t r a s t , an am m onium s u l f a t e f r a c ­
tio n o f th e S-100 s u p e r n a t a n t a lo n e s t i m u l a t e d th e p o l y c y t i d y l i c a c id
■7
77
d i r e c t e d s y s t e m . S in c e a h ig h s a l t r ib o s o m a l w a s h f r a c tio n w a s know n
to c o n ta i n th e th r e e b a c t e r i a l p ro te in i n i t i a t i o n fa c to r s F I, FII a n d F ill
(D u b n o ff & M a i t r a , 1969; O c h o a , 19 69; R e v e l, b e l o n g , Braw erm an &
G r o s , 19 68; R e v e l, H e r z b e r g , B e c a ra v ic & G r o s , 19 68), i t w a s p o s s i b l e
t h a t CAR's a p p a r e n t i n h ib iti o n o f i n i t i a t i o n in th e MS2 mRNA a n d p o l y c y t i d y l i c a c i d d i r e c t e d s y s t e m s w a s r e l a t e d to th e f u n c tio n o f t h e s e
f a c t o r s . The s t im u la to r y e f f e c t o f th e r ib o s o m a l w a s h f a c t o r s , n o rm a lly
o n ly s e e n w ith th e n a t u r a l mRNA s y s t e m , w o u ld s u g g e s t t h a t i n i t i a t i o n
in th e p o l y c y t i d y l i c a c i d d i r e c t e d s y s te m w a s s im ila r to i n i t i a t i o n in
th e MS2 s y s t e m .
.
.
.
A s e r i e s o f e x p e r im e n t s w a s r e q u ir e d to b rid g e th e g ap b e tw e e n
t h e g e n e r a l h y p o t h e s i s o f i n h ib iti o n o f i n i t i a t i o n in t h e MS2 s y s te m by
CAP, a n d th e d e t a i l e d r o l e s , i f a n y , o f th e r e c o g n i z e d b a c t e r i a l i n i ­
tia tio n fa c to rs .
An MS2 mRNA d i r e c t e d in v itr o p r o te in s y n t h e s i z i n g s y s te m w a s
d e v e lo p e d w h ic h w a s h ig h ly d e p e n d e n t u p o n a d d e d r ib o s o m a l w a s h f a c ­
to rs.
The CAP s e n s i t i v i t y o f t h i s s y s t e m w a s found to b e o v e r tw ic e a s
s e n s i t i v e to CAP a s th e S - 3 0 a s s i s t e d s y s t e m . F ig . 2 4 . This i n c r e a s e d
s e n s i t i v i t y to CAP w a s h ig h ly s u g g e s t i v e o f a n i n t e r a c t i o n b e tw e e n th e
drug a n d so m e fa c to r(s ) p r e s e n t in t h e h ig h s a l t r ib o s o m a l w a s h .
A p u rif ie d s y s t e m w a s n e e d e d to d if f e r e n tia te p r o te in s y n t h e s i s
w h ic h h a d a n a b s o l u t e r e q u ire m e n t for r ib o s o m a l w a s h f a c t o r s from p r o ­
t e i n s y n t h e s i s w h ic h o c c u r r e d in th e a b s e n c e o f t h e s e f a c t o r s . By
78
s u b j e c t i n g a p r e - i n c u b a t e d in v itro MS'2 d i r e c t e d s y s te m to c e n t r i f u g a ­
tio n a t 105,000 x g for tw o h o u r s , th e r ib o s o m a l c o m p le x c o u ld b e p e l l e t ­
e d o n to a 10% s u c r o s e p a d , c o l l e c t e d a n d p l a c e d in to n e w in c u b a t io n
m i x t u r e , c o m p le te w ith c ru d e i n i t i a t i o n f a c t o r s . A s i g n i f i c a n t am o u n t of
- a m in o a c i d s w a s i n c o r p o r a t e d .
The p H 5 ’ f r a c tio n a lo n e w o u ld n o t s tim u la te th e r ib o s o m a l c o m ­
p l e x r e c o v e r e d b y c e n t r i f u g a t i o n . It w a s c o n c lu d e d t h a t th e s tim u la tio n
b y r ib o s o m a l w a s h f a c t o r s r e s u l t e d from t h e i r re q u ir e m e n t for r e ­
i n i t i a t i o n o f H1RNA. W h e n rib o s o m a l w a s h f a c to r s w e re n o t a d d e d to th e
c e n tr if u g e d c o m p l e x e s , th e s y s t e m w o u ld n o t in c o r p o r a te
14
C -a m in o
a c i d s d u e to re m o v a l o f i n i t i a t i o n f a c to r s b y th e c e n t r i f u g a t i o n .
By t h i s
m e a n s i t w a s p o s s i b l e t h a t a ^ C - a m i n o a c i d in c o r p o r a tio n in v o lv in g
i n i t i a t i o n a lo n e c o u ld b e d i s t i n g u i s h e d from o n e in v o lv in g b o th in i t i a t i o n
a n d e l o n g a t i o n . It w a s n o w p o s s i b l e to t e s t GAP's e f f e c t bn c ru d e i n i ­
t i a t i o n f a c to r d e p e n d e n t a n d c ru d e i n i t i a t i o n f a c to r i n d e p e n d e n t incor--.
14
.
■
p o r a tio n o f
C -a m in o a c i d s .
It w a s fo u n d t h a t CAP h a d a s i g n i f i c a n t l y g r e a t e r e f f e c t on
14
C-
a m in o a c i d in c o r p o r a tio n s p e c i f i c a l l y c a t a l y z e d b y th e rib o s o m a l w a s h
f a c t o r s , p ro d u c in g a 45% i n h i b i t i o n . R e s id u a l
14
C - a m in o a c i d in c o rp o ra ­
tio n w ith ' c ru d e i n i t i a t i o n f a c to r s a b s e n t w a s n o t a f f e c t e d (F ig . 2 7 ).
T h e s e r e s u l t s s u g g e s t t h a t CAP e x e r t s i t s e f f e c t d u rin g i n i t i a t i o n . After
i n i t i a t i o n , i t e x e r t s a , s i g n i f i c a n t l y lo w e r e f f e c t . The m e th o d o lo g y
u t i l i z e d to d i f f e r e n t i a t e i n i t i a t i o n a n d e lo n g a t io n d e p e n d e n t
14
C -a m in o
79
a c i d in c o r p o r a tio n inv o lv ed , th e s e l e c t i v e p r e s e n c e or a b s e n c e o f c ru d e
in itia tio n fa c to rs.
This s u g g e s t s t h a t i n i t i a t i o n c a t a l y z e d b y th e i n i ­
t i a t i o n f a c to r s th e m s e lv e s is i n h i b i t e d b y CAP.
The w ork p r e s e n t e d h e re th e r e f o r e ru le s o u t m o d e ls o f p re m a tu re
r e l e a s e , random i n i t i a t i o n , i n h ib iti o n o f te r m in a tio n a n d in h ib iti o n of
e lo n g a t io n a s th e m e c h a n is m o f a c t i o n o f CAP a t lo w c o n c e n t r a t i o n s in
th e MS2 mRNA d i r e c t e d s y s t e m . In a d d i t i o n , th e d a ta s tr o n g ly a rg u e in
fa v o r o f a m o d el w h e r e b y CAP i n h i b i t s t h e i n i t i a t i o n o f p o ly p e p tid e s y n ­
t h e s i s s p e c i f i c a l l y . c a t a l y z e d by th e r e c o g n iz e d b a c t e r i a l i n i t i a t i o n
fa c to rs.
• --
.
80
T a b le I
P r o p e r tie s o f th e E. c o ll i n i t i a t i o n fa c to r s
D e s ig n a tio n
M o l e c u l a r W e ig h t ( W a h b a 7 1969)
A. . . fr . . FI
10,000
B ;. . f 3 . . . FII
2 0,000
C . . . f 2 . . . F ill
80,000
A. . B. . 0 . .
nc
n o ta tio n ( O c h o a , 19 69)
fr • f ^ • 6f ^ • •
FI. .F I I . . F i l l .
81
Inlerm ediote I ?
‘ACCOM M O OATlON-
•A C C O M M O D A T IO N *
s T t A N S lO C A T lO N
F ig . I .
1
S c h e m a tic r e p r e s e n t a t i o n o f th e e v e n t s d u rin g p ro te in
s y n t h e s i s ( a f te r T h a c h & T h a c h 1 1 9 7 1 ).
82
F ig . 2 .
Cl
Cl
O = C CH9 OH
I i 2
N-C-H
H I
H— C - O H
NO2
STRUCTURE'OF CHLORAMPHENICOL*.
•D H th r e o -E -D I C H L O R O A C E T A M ID O -l-p -N IT R O P H E N Y L 1 , 3-P R O PA N E D IO L
83
1 2
3
4
5
6
8
7
9
10
11
12
13
14
15
I ALA SER ASN PHE THR GLN PHE VAL LEU VAL ASN ASP GLY GLY THR
16 GLY ASN VAL THR VAL ALA PRO SER ASN PHE ALA ASN GLY VAL ALA
31 GLU TRP ILU SER SER ASN SER ARG SER GLN ALA TYR LYS VAL THR
46 CYS SER VAL ARG GLN SER SER ALA GLN ASN ARG LYS TYR THR ILU
61 LYS VAL GLU VAL PRO LYS VAL ALA THR GLN THR VAL GLY GLY VAL
76 GLU LEU PRO VAL ALA ALA TRP ARG SER TYR LEU ASN MET GLU LEU
91 THR ILU PRO ILU PHE ALA THR ASN SER ASP CYS GLU LEU ILU VAL
106 LYS ALA MET GLN GLY LEU LEU LYS ASP GLY ASN PRO ILU PRO SER
121 ALA ILU ALA ALA ASN SER GLY ILU TYR
COMPOSITION
14
4
6
4
13
2
9
7
6
9
ALA
PHE
LYS
TYR
SER
CYS
GLY
LEU
PRO
THR
3
0
2
6
I.4
ASP
HIS
MET
GLN
VAL
5
8
11
4
2
GLU
ILU
ASN
ARG
TRP
TOTAL NO, OF ACIDS = 129
F ig . 3 .
Amino a c i d s e q u e n c e o f MS2 v iru s c o a t p r o te in (W e b er &
K o n ig s b e r g , 19 67).
,
f
-
. : T - r . / — C".. ; Vf:- -
" C -"I- —— -
'
/M ..
84
UV s p e c tru m o f h ig h ly p u rif ie d MS2 v i r u s .
OPTICAL DENSITY
F ig . 4 .
WAVE LENGTH IN MILLI MICRONS
--.A
85
in fect MS-2
m .o .i . =3
grow 10 hours
centrifuge
c e ll d eb ris
5,000 xg for 30 min
E. coli Q -13
grown in TYS-media
to 10® c e lls
14 lite rs
A
add 311 g/1
ammonium su lfa te
le t s i t overnight
A
p lace su p ern atan t
in c h illed carboy
centrifuge p recip itate
5,000 xg for 30 min.
allow p recip itate to
d isso lv e in
pH 8 d is tille d w ater
centrifuge
5,000 xg for 30 min.
to clarify
J
su sp en d in
pH 8 d is t. w ater
centrifuge
80,000 xg for 3 hours
to p e lle t phage
b ottle and
store a t -9 0°C
Yield = 10 mg
T iter = 10*5 v iru s/m l
G R O W T H A N D P U R I F I C A T I O N OF M S -2 V I R U S
F ig . 5.
Flow d ia g ra m i l l u s t r a t i n g th e g ro w th a n d p u r if ic a tio n of
MS2 v i r u s . D e t a i l s a re d e s c r i b e d in " M a t e r i a l s and M e th o d s " .
8 6
add 0.1 ml
2 M ammonium carbonate
shake 10 min^
centrifuge 5 min.
add 1.0 ml phenol
room tem p.
3,000 xg
1.0 ml
phage
su sp en sio n
blow off
eth er with
nitrogen
e x tract 6 tim es
w ith cold eth er
aqueous phase
sep arated
aqueous pi e
added to orig in al
aqueous p h ase
add 6 drops cold
3 M sodium a c e ta te
phenol
phase
retained
ex tra c t phenol with
1.0 ml d is tille d w ater
V
VIRAL COAL PROTEIN
add 2 volum es cold
a b so lu te ethanol
add sodium a c e ta te
until turbid
centrifuge 5 min.
3,000 xg
allow to flo ccu late
in cold
U
D
VIRAL RNA
d is so lv e in
d is tille d w ater
store at -9 0 ° C
lyophyll7.e to
d ry n ess
v
Yield = 50% of virus
E X T R A C T I O N OF M S -2 m - R N A A N D C O A T P R O T E I N
F ig . 6.
Flow d ia g ra m i l l u s t r a t i n g th e e x tr a c ti o n o f MS2 RNA and
c o a t p ro te in from th e b a c te r io p h a g e M S 2 . D e t a i l s a re g iv e n in
" M a t e r i a l s a n d M e th o d s " .
87
E
u
O
CO
C hrom atography-
Fig . 7. D iag ram o f th e d im e n s io n s o f an MS2 f i n g e r p r in t. E le c tr o ­
p h o r e s i s w a s p e rfo rm ed in th e i n d i c a t e d d ir e c tio n a t a v o l t a g e flu x of
30y/cm. for 2.5 h o u r s . After a ir d ry in g o v e r n ig h t, p o in ts A-A a n d B-B were
c o n n e c te d b y p l a s t i c p a p e r c lip s , form ing a c y lin d e r. C h ro m a to g ra p h y was
th e n a llo w e d to e n s u e for 8 h o u rs a f t e r 12 h ours of e q u i l i b r a t i o n w ith th e
c h o s e n b u ffe r.
88
F ig . 8 .
E ffe c t o f Mg+ + c o n c e n t r a t i o n on S -3 0 i n c o r p o r a t i o n s .
O n ly th e Mg+ + c o n c e n t r a t i o n w a s v a r ie d by u s in g a n LMSN b a s e b u ff e r.
The i n c u b a t io n tim e w a s 40 m i n u t e s . O ne h u n d re d m ic ro g ra m s o f MS2
RNA w a s a d d e d to e a c h s a m p l e . The r a d i o a c t i v e c o u n tin g te c h n i q u e s
a n d th e c o n c e n t r a t i o n o f r e a g e n t s a re d e s c r i b e d in " M a t e r i a l s and
M e th o d s " .
i!
90
F ig . 9 .
K in e tic s o f ^ C - a m i n o a c id in c o r p o r a tio n . O ne h u n d red
m ic ro g ra m s o f MS2 mRNA w e re a d d e d to e a c h s a m p le c o n ta i n in g th e
r e a g e n t s a s d e s c r i b e d in " M a t e r i a l s a n d M e th o d s " . In c o rp o ra tio n w a s
te r m in a te d a t th e a p p r o p r ia te tim e b y th e a d d itio n o f 6 m l. o f 5% t r i ­
c h lo ro a ce tic a c id .
The e n s u in g s a m p le p r e p a r a tio n a n d r a d i o a c t i v e
c o u n tin g t e c h n i q u e a r e d e s c r i b e d in " M a t e r i a l s a n d M e th o d s " .
I
MINUTES
92
F ig . 10.
S tim u la to ry e f f e c t o f MS2 m e s s e n g e r RNA. V arying
a m o u n ts o f MS2 RNA w e re a d d e d to a n S -3 0 a s s i s t e d p r o te in s y n t h e ­
s i z i n g s y s t e m . A Mg++ c o n c e n tr a tio n o f 8.5 m M , an i n c u b a t io n tim e o f
40 m i n u t e s , a n d a te m p e r a tu r e o f 3 7 ° C w e re u s e d . F u rth e r d e t a i l s o f th e
in c u b a t io n a re d e s c r i b e d in " M a t e r i a l s a n d M e th o d s " .
IOO
/ig M S 2
120
RNA / ml.
140
160
180
2 0 0
94
F ig . 11.
D e p e n d e n c e o f in c o r p o r a tio n on a d d e d
14
C - a m in o a c i d s .
++
The fin a l in c u b a t io n v o lu m e w a s 1.0 m l . , a n d th e Mg
c o n c e n tr a tio n
w a s 8.5 mM in a ll c a s e s . V arying a m o u n ts o f
14
14
C -Iy s in e and C -
a r g in in e w e re a d d e d to th e in c u b a t io n t u b e s , w ith th e v o lu m e s a d ­
j u s t e d by th e a d d itio n o f b u ffe r L M S N . All o th e r d e t a i l s a re d e s c r i b e d
in " M a t e r i a l s a n d M e th o d s " .
I
ftCi of 1^C LYSINE AND
C ARGININE / m l .
96
F ig . 12.
c o u n ts.
E ffe ct of c h lo r a m p h e n ic o l on t o ta l a c i d p r e c i p i t a b l e
The in c u b a t io n v o lu m e s w e re in a ll c a s e s 1 .0 m l., w ith an
in c u b a t i o n tim e o f 40 m in u te s a n d a te m p e r a tu r e o f 3 7 ° C . V arying
a m o u n ts o f c h lo r a m p h e n ic o l w e re a d d e d from a s to c k s o lu tio n of
1.0 m g. c h lo r a m p h e n ic o l p e r 10 m l. b u ffe r L M SN . All o th e r d e t a i l s a re
d e s c r i b e d in " M a t e r i a l s a n d M e th o d s " .
I
.
I
Z
0
hQQ
1
Z
LO
"4
O
2
4
6
8
CHLORAMPHENICOL / m l .
I
12
14
98
F ig . 13.
E ffect o f c h lo r a m p h e n ic o l on p e p t i d e s r e l e a s e d in v i t r o .
The fin a l in c u b a t io n v o lu m e w a s in a l l c a s e s 2.0 m l . The s a m p l e s w e re
i n c u b a t e d for th e i n d i c a t e d tim e a t 3 7 ° C a n d c h i l l e d to 0 ° C in an i c e /
s a l t b a th a t th e e n d o f th e in c u b a t i o n . S a m p le s w e re c e n tr if u g e d a t
105.000 xg for 2 h o u rs a s d e s c r i b e d in " M a t e r i a l s a n d M e th o d s " . At th e
c o n c l u s i o n o f t h i s c e n t r i f u g a t i o n , th e s u p e r n a t a n t w a s re m o v e d a n d
3.0
14
m g. ly s o z y m e a d d e d a s a p ro te in c a r r i e r . The s u p e r n a t a n t
C p ro ­
d u c ts w e re th e n p r e c i p i t a t e d w ith 5% t r i c h l o r o a c e t i c a c i d a n d r a d io ­
a c t i v i t y d e te r m in e d a s d e s c r i b e d in " M a t e r i a l s a n d M e th o d s " .
-r-r
4
ro
O
—x
3 - C h lo r a m p h e n ic o l
UJ
H
D
cr
LU
CL
(/)
/ C h lo r a m p h e n ic o l
IZ
2 . 5 /x g /m l.
z>
O
O
IO
20
MINUTES
Released Peptides
1 0 0
F ig . 14.
P e p tid e map o f n a t i v e MS2 c o a t p r o t e i n . T hree m g. of
p u rif ie d n a tiv e MS2 c o a t p ro te in w e re o x id iz e d a n d d i g e s t e d w ith
T P C K -tre a te d t r y p s i n .
The r e s u l t i n g d rie d p e p t i d e s w e re s u b j e c t e d to
e l e c t r o p h o r e s i s in b u f f e r EBN-I on W h atm an N o . 3mm f i l t e r p a p e r . After
a i r d ry in g th e e l e c t r o p h o r e t o g r a m , c h ro m a to g ra p h y w a s perfo rm ed for
8 to 10 h o u rs in b u ffe r CBN- I .
c o m p le te d e t a i l s .
Refer to " M a t e r i a l s a n d M e th o d s " for
101
7
UJ H
O O 4> M O
O Origin
PEPTIDE n NGERPRINT OF NATIUF MS-?
COflT PROTEIN
Chromatography
102
F ig . 15.
P e p tid e map o f th e in v itr o MS2 d i r e c t e d r e l e a s e d p e p ­
t i d e s . A s ta n d a r d 2.0 m l. in c u b a t io n m ix tu re w a s in c u b a t e d for 40 m in ­
u t e s a t 3 7 ° C . The s a m p le w a s c e n tr if u g e d for tw o h o u rs a t 105,000 xg
a n d t h e s u p e r n a t a n t w a s f i n g e r p r in te d . Three m g. o f ly s o z y m e w e re
a d d e d a s a p r o te in c a r r i e r a n d o x i d a t i o n , d i g e s t i o n , a n d fin g e rp r in tin g
c a r r i e d o u t a s d e s c r i b e d in " M a t e r i a l s a n d M e th o d s " . The fin g e rp r in t
w a s e x p o s e d to Kodak 8 " x lO " X -ra y film for 10 d a y s a n d d e v e l o p e d . A
t r a c i n g w a s t h e n m ad e o f th e r a d i o - o p a q u e s p o ts a s s h o w n h e r e .
/
103
-N
,
I
UlH
O O +» *4 O
11
O M O t t H m
RELEASED M
ATERIAL
FINGERPRINT OF THE
IN VITRO PEPTIDES
—
No Chloramphenicol-
6
V ____
Origin
Chromatography-----------------------
y
104
F ig . 16.
E ffe c t o f c h lo r a m p h e n ic o l on th e p e p t i d e m ap o f p e p t i d e s
r e l e a s e d in v i t r o . A s t a n d a r d 2.0 m l. i n c u b a t io n m ix tu re w a s i n c u b a t e d
for 40 m in u te s a t 3 7 ° C in th e p r e s e n c e o f 2.5 p g ./m l. o f c h lo r a m p h e n i c o l.
The p ro c e d u re a f te r i n c u b a t io n w a s i d e n t i c a l to t h a t o f F ig . 15, a n d a
s t a n d a r d fin g e r p r in t w a s m ade o f t h e 105,000x g s u p e r n a t a n t . D e t a i l s o f
th e th r e e p e p tid e m ap s a r e d e s c r i b e d in " M a t e r i a l s a n d M e th o d s " .
-V •
105
CI
O O
O
O
co
O
O OO
O
12
•With ChloramphenicolJ 2.5ug/ml*
RELCASED PEPTIDES
Q Origin
FINGERPRINT OF THE C1 4 IN UITRO PEPTIDES
Chromatography
F ig . 17.
E ffe ct o f c h lo r a m p h e n ic o l on a c id p r e c i p i t a b l e c o u n ts o f
th e r ib o s o m a l b o u n d in v itr o p e p t i d e s .
The i n c u b a t io n p ro c e d u re w a s
i d e n t i c a l to t h o s e for F ig . 16, e x c e p t th e s u p e r n a t a n t w a s rem o v ed from
th e 105,000 xg c e n t r i f u g a t i o n , a n d th e p r e c i p i t a t e w a s s o l u b i l i z e d in
2 .0 m l. o f b u f f e r L M S N . Three m g. o f ly s o z y m e w a s th e n a d d e d a n d th e
a c i d p r e c i p i t a b l e r a d i o a c t i v i t y d e te r m in e d a s d e s c r i b e d in " M a t e r i a l s
a n d M e th o d s " .
~ V ' ' vo .~ r.. ■
■
107
-
Chloramphenicol
+Chloramphenicol
2 .5 /ag /m l.
O
IO
MINUTES
.Ri b o so m a I Bound Material
Va..
108
F ig . 18.
P e p tid e m ap o f th e in v itr o MS2 d i r e c t e d r ib o s o m a l
b o u n d p e p t i d e s . A s t a n d a r d 2.0 m l. in c u b a t io n w a s c e n tr if u g e d a t
105,000 x g for 2 h o u rs a n d th e s u p e r n a t a n t re m o v e d . The p r e c i p i t a t e w a s
s o l u b i l i z e d in 2.0 m l. o f b u ffe r LMSN a n d 3.0 m g. o f ly s o z y m e a d d e d a s a
p r o te in c a r r i e r . The s o l u t i o n w a s th e n t r e a t e d a s d e s c r i b e d in
" M a t e r i a l s a n d M e th o d s " , a n d a s t a n d a r d p e p tid e map w a s m a d e .
'
--
109
\
I
UIH
O O t i H O
CL. C O h O H H O
RIBOSOBlflL BOUND
r i NOBRPRINT OF THE C14 IN VITRO PEPTIDES
(Size of circle approximates density)
-No Chloramphonlcol-
Q
Origin
Chromatography ------------------
J
F ig . 19.
E ffe c t o f c h lo r a m p h e n ic o l on th e p e p tid e map o f th e
rib o s o m a l b o u n d in v itr o p e p t i d e s . A s ta n d a r d 2.0 m l . in c u b a t io n m ix ­
tu re w a s i n c u b a t e d for 40 m in u te s a t 3 7 ° C in th e p r e s e n c e o f 2.5 Mg./m l .
o f c h l o r a m p h e n i c o l . The s a m p le w a s c e n tr if u g e d for tw o h o u rs a t
105,000 x g a n d a s t a n d a r d fin g e r p r in t w a s m ade o f th e m a te r ia l p r e c i p i ­
t a t e d a t 105,000x g . The d e t a i l s o f th e p ro c e d u re a re s u m m a riz e d in
" M a t e r i a l s a n d M e th o d s " . The c o m p le te d fin g e rp r in t w a s e x p o s e d for
10 d a y s to Kodak X - r a y film , a n d u p o n d e v e lo p m e n t o f t h e a u to r a d io g r a m ,
a t r a c i n g w a s m ad e o f t h e r a d i o - o p a q u e s p o t s .
* • •
-
\
r
"-T
• ■
—
111
r
x
o
>3'
x
O
O
6
O
7
O
M- to O M O 3 " 0
O M M - O O M M
5
11
RIBOSOHlftL BOUND
FINGERPRINT OF THE.
s
@ Origin
V
I_N VITRO PEPTIDES
—
With Chloramphenicol; 2.5ug/ml—
Chromatography
y
112
\
F ig . 2 0 .
Q u a n tita tiv e e f f e c t o f c h lo r a m p h e n ic o l on t h e rib o s o m a l
b o u n d in v itro tr y p t ic p e p t i d e s . E a c h o f th e fin g e r p r in ts from F i g s . 18
a n d 19 w e re m a tc h e d to t h e i r r e s p e c t i v e a u to r a d io g r a p h . U s in g a s o f t
l e a d p e n c i l , t h e a r e a s on th e fin g e r p r in ts w e re m ark ed c o r r e s p o n d in g to
th e r a d i o - o p a q u e a r e a s on th e a u to r a d io g r a p h . T h e s e a r e a s on th e
fin g e r p r in t w e re c u t o u t a n d th e r a d i o a c t i v i t y d e te r m in e d b y liq u id
s c i n t i l l a t i o n c o u n tin g . T h is m e th o d o lo g y i s d e s c r i b e d in d e t a i l in
" M a t e r i a l s a n d M e th o d s " . The p o in ts p l o t t e d r e p r e s e n t th e r a d io a c ti v e
l e v e l o f th e c u t o u t fin g e r p r in t s p o t m in u s a c o n tro l s p o t from th e e d g e
o f th e s a m e f in g e r p r in t.
/
\
COUNTS PER MINUTE (xIO"
113
- C h lo r a m p h e n ic o l
f C h lo r a m p h e n ic o l
2 . 5 / llq / m L
PEPTIDE NUMBER
I
114
F ig . 21. , Mg
t i t r a t i o n c u r v e .o f th e p u rif ie d in v itr o s y s t e m .
The
c o n c e n t r a t i o n o f Mg++ w a s v a r ie d o v e r th e ra n g e i n d i c a t e d a n d th e
0 . 5 - m l . s a m p le w a s i n c u b a t e d a t 3 7 ° C for 40 m i n u t e s . S ta n d a rd c o n c e n ­
tr a tio n s of a l l r e a g e n t s w a s u s e d .
T o tal a c i d p r e c i p i t a b l e r a d i o a c t i v i t y
w a s d e te r m in e d a s d e s c r i b e d in " M a t e r i a l s a n d M e th o d s " .
I,
M agn esiu m Io n T itration C u rv e— P u rified S ystem
C o n c e n tr a tio n of M agn esiu m I o n .. . mM
116
F ig . 2 2 .
I
T itra tio n c u rv e o f c ru d e i n i t i a t i o n f a c to r s a g a i n s t th e
p u r if ie d s y s t e m . The a m o u n ts o f c ru d e i n i t i a t i o n f a c to r s v a r ie d a s d e ­
s c r i b e d . A s ta n d a r d in c u b a t i o n v o lu m e o f 0.5 m l. w a s u s e d . I n c u b a tio n
w a s for a d u ra tio n o f 30 m in u te s a t 3 7 ° C .
C o n c e n tr a tio n s o f a l l o th e r
i n c u b a t i o n r e a g e n t s w e re h e ld c o n s t a n t . R a d io a c tiv e c o u n tin g i s d e ­
s c r i b e d in " M a t e r i a l s a n d M e th o d s " .
I
v-
In itia tio n Factor T itration C u rve— P urified S ystem
‘
\
A280 U n its 0^ c r u d e In itia tio n Factors
118
/
F ig . 2 3 .
K in e tic s o f ^ C - a m i n o a c i d in c o r p o r a tio n o f th e p u rif ie d
s y s t e m . S ta n d a rd 0 .5 -m l. i n c u b a t io n m ix tu r e s w e re i n c u b a t e d for th e
i n d i c a t e d t i m e s , a n d 5% t r i c h l o r o a c e t i c a c i d a d d e d to te r m in a te i n c o r ­
p o r a tio n . The s a m p l e s w e re th e n c o u n te d a s d e s c r i b e d in " M a t e r i a l s
a n d M e th o d s " .
- __ -T -
3: xj o
K in etics of C A m in o A cid In co rp o ra tio n — P urified S ystem
Time (m inutes)
V
12 0
F ig . 2 4 .
E f f e c t o f c h l o r a m p h e n i c o l on t o t a l a c i d p r e c i p i t a b l e
counts of the purified s y ste m .
S t a n d a r d i n c u b a t i o n s a m p l e s o f 0.5 ml.
w e r e i n c u b a t e d for 40 m i n u t e s a t 3 7 ° C w i t h t h e i n d i c a t e d c o n c e n t r a t i o n
of c h l o r a m p h e n i c o l .
and M ethods".
R a d i o a c t i v e c o u n t i n g i s d e s c r i b e d in " M a t e r i a l s
. •
I
S-oo
C h lo ra m p n en ico l In h ib itio n C u rv e— P u rified S ystem
C o n cen tra tio n of C h Ioram pn enicol (x IO ) mM
I
122
F ig . 2 5 .
E f fe c t o f c r u d e i n i t i a t i o n f a c t o r s on a p u r i f i e d s y s t e m
re-in cu b ated after a 2-hour centrifugation.
S t a n d a r d c o m p l e t e 1.0-ml.
m i x t u r e s w e r e i n c u b a t e d for 4 m i n u t e s a t 3 7 ° C , a n d c h i l l e d t o 0 ° C in an
i c e / s a l t bath.
The s a m p l e s w e r e c a r e f u l l y l o a d e d i n t o Beckman 2 .0 - m l .
n i t r o c e l l u l o s e c e n t r i f u g e t u b e s , c o n t a i n i n g a 0.2 - m l . p a d o f 10% s u c r o s e
in b u ff e r L M S N - 1 6 , b r i n g i n g t h e fi n a l v o l u m e to 2.0 m l . w i t h b uff er
LMS N-16. After c e n t r i f u g a t i o n a t 105,000 x g for 2 h ours in t h e Beckman
M o d e l L , t h e s u p e r n a t a n t w a s c a r e f u l l y r e m o v e d l e a v i n g a t o t a l volu m e
in t h e c e n t r i f u g e t u b e of 0.3 m l. This 0 .3 - m l . s u c r o s e p a d w a s a d d e d to
a c h i l l e d 1.0-ml. i n c u b a t i o n mixtu re m in u s r i b o s o m e s a n d mRNA. In
a d d i t i o n , c r u d e i n i t i a t i o n f a c t o r s w e r e a d d e d to one s a m p l e . After m i x ­
i n g , t h e s a m p l e s w e r e i n c u b a t e d a t 3 7 ° C for 25 m i n u t e s a n d t h e r a d i o ­
a c t i v i t y d e t e r m i n e d a s d e s c r i b e d in " M a t e r i a l s a nd M e t h o d s " .
» J- •
V
-
.
:
—
a
. 1 - '• . - . . - z ?
r._
I
E ffect of In itia tio n Factors on a P urified S ystem R e-started Up A fter a Two H our S p in
3
123
o
+ In itia tio n Factors
___
(209)
- In itia tio n Factors
100 -
C en trifu g a tio n at O'C
Time (m inutes)
124
Fig. 26.
D e p e n d e n c e of t h e p u r i f i e d s y s t e m on t h e pH 5' frac tio n.
The r e - i n c u b a t i o n m i x t u r e s c o n t a i n e d a l l i n g r e d i e n t s a s a s t a n d a r d
s a m p l e , e x c e p t t h a t in o n e c a s e t h e pH 5' f r a c t i o n w a s d e l e t e d .
The
s a m p l e s w e r e r e m o v e d a t t h e e n d of t h e . 2 5 - m i n u t e r e - i n c u b a t i o n a n d
t e r m i n a t e d w i t h 5% t r i c h l o r o a c e t i c a c i d .
Radioactive counting was as
d e sc rib e d /in " M aterials and Methods".
►
\
; rr
-_ =. - - r -
D ep end ance of P u rified S ystem on tn e pH 5 F raction
^ (578)
+ In itia tio n Factors
+ Ph 5 Fraction
OQ-S
12 5
A (146)
+ In itia tio n Factors
- Ph 5 Fraction
C en trifu g a tio n at O1C for 2 h o u r s
Time (m inutes)
126
1800
1600
1400
1200
1000
800
C
P
M 600
400
200
0
I
0
10
C oncentration of chloramphenicol
(pig./ml.)
''
'
= c r u d e i n i t i a t i o n f a c t o r s p r e s e n t ( dur in g r e - i n c u b a t i o n )
= c r u d e i n i t i a t i o n f a c t o r s a b s e n t ( dur in g r e - i n c u b a t i o n )
Ef fe ct of c h l o r a m p h e n i c o l on i n i t i a t i o n f a c t o r d e p e n d e n t
a n d i n d e p e n d e n t i n c o r p o r a t i o n of 14C - a m i n o a c i d s .
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