Mechanism of action of chloramphenicol : effect on T4D head coat protein synthesis
by Charles Edward Green
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in Genetics
Montana State University
© Copyright by Charles Edward Green (1970)
Abstract:
Inhibition of protein synthesis by chloramphenicol (CAP) was completely reversible on bacteriophage
T4 mRNA, nascent peptides, and host E. coli ribosomes after removal of CAP. This was established by
using T4D w.t. and T4 amber mutants amB17 and amN54 with density gradient polysome analysis and
polyacrylamide disc gel electrophoresis.
The mRNA continues to code for protein synthesis after transient treatment with CAP. This finding
was demonstrated by first treating T4 infected cells with rifampicin to prevent further mRNA synthesis,
which was followed by treatment with CAP to inhibit protein synthesis. The CAP was subsequently
removed and the utilization of mRNA which had been formed prior to rifampicin treatment was then
tested by observing the rate of C^14-arginine incorporation. This incorporation increased steadily
during the first 10 minutes after CAP removal, but 20 minutes later its rate had declined only slightly.
This result indicated that CAP treated mRNA remained functional and continued to code for protein
synthesis after removal of the antibiotic.
Messenger RNA-ribosome complexes which are subjected to CAP treatment do resume protein
synthesis after removal of the antibiotic. Nascent peptides labeled with C^14-leucine were trapped in
the polysome region of a sucrose density gradient if CAP was added to T4 infected cultures. With these
cells C^14-leucine could be chased out of the polysome region when C^l2-leucine was added after
CAP was removed. Eighty-five percent of this label was removed from this region in a 10 minute chase
indicating that ribosomal movement and function have been restored.
Nascent peptides are completed and form normal head coat protein after transient treatment with CAP.
This was demonstrated on polyacrylamide gels by the appearance of the head coat protein band when
labeled nascent peptides in CAP treated cells were completed after the removal of CAP to form normal
head coat protein.
This was demonstrated by a second method: incomplete head coat peptides of cells transiently treated
with CAP were chased into the protein aggregate region of polyacrylamide gels . Therefore, nascent
peptides in CAP treated cultures are completed and released normally after removal of the antibiotic. MECHANISM OF ACTION OF CHLORAMPHENICOL:
EFFECT ON T4D HEAD COAT PROTEIN SYNTHESIS
by
CHARLES EDWARD GREEN, JR.
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 lty in p a r tia l
fu lfillm e n t o f th e re q u ire m e n ts for th e d e g re e
of.
DOCTOR OF PHILOSOPHY
. in
G e n e tic s
A pproved:
H e a d , M ajo r D e p a rtm e n t
MONTANA STATE UNIVERSITY
B o zem an , M o n ta n a
J u n e , 1970
iii
ACKNOWLEDGEMENTS
The a u th o r th a n k s :
DR. GORDON R.. JULIAN fo r h is p a tie n t g u id a n c e , u n d e rs ta n d in g ,
a n d c o n s tr u c tiv e c r itic is m th ro u g h o u t th is s tu d y .
DR. SAMUEL J . ROGERS fo r h is in t e l l e c t u a l e n c o u ra g e m e n t an d
h is a s s i s t a n c e in m a n u s c rip t p r e p a r a tio n .
DR. PALMER D . SKAAR for h is a d v ic e an d a s s i s t a n c e in m an u ­
s c r ip t p r e p a r a tio n .
DR. HELEN J . CAMERON fo r h e r c o n tin u e d i n t e r e s t a n d .h e lp fu l
d is c u s s io n .
DOROTHY SPAIN GREEN fo r h e r u n w a v e rin g s u p p o rt an d e n c o u r­
a g e m e n t d u rin g th is s tu d y .
The N a tio n a l I n s titu te s o f H e a lth fo r P re d o c to ra l F e llo w s h ip
5 FOI G M 3 8 3 1 4 .
. TABLE OF CONTENTS
PAGE
INTRODUCTION
C h lo r a m p h e n ic o l........................................................................................
I
R a t i o n a l .........................................................................................................
4
MATERIALS AND M E T H O D S ................................................. .............................-
7
B a c te ria l an d B a c te rio p h a g e S t r a i n s ......................................
7
M ed ia an d S u p p l e m e n t s ................................
7
B a c te ria l an d B a c te rio p h a g e S to ck P r e p a r a tio n ..............................
9
B a c te rio p h a g e A s s a y ...............................................................................
10
G row th T e m p e r a t u r e s ...............................................................................
10
O ne S tep G row th C u r v e s ........................................................................
11
B io c h e m ic a ls , R e a g e n ts , an d S u p p lie s
11
R a d io is o to p e s
..................
............................................................................................
12
B uffers . . .....................................................................................................
12
R ib o n u c le a s e R e m o v a l ..................................................................... ... .
13
G e n e ra l E x p e rim e n ta l P r o c e d u r e ........................................................
14
C e ll L y s is P r o c e d u r e ................................................
15
R ib o n u c le a s e T r e a t m e n t .......................................
16
S u c ro s e D e n s ity G ra d ie n t P r o c e d u r e .................................... ... . ..
16 .
P r e c ip ita tio n an d P la tin g P ro ce d u re for-D .ensity
G ra d ie n t F r a c t i o n s ....................... ................................ •.....................
17
V
MATERIALS AND METHODS (C o n tin u e d )
A c id ic P o ly a c ry la m id e U rea D is c G e l E le c tro p h o re s is . . . .
18
L iq u id S c in tilla tio n C o u n tin g o f P o ly a c ry la m id e G e ls
....
18
.....................................................................
19
T e rm in o lo g y . ....................... ... .................................................................
19
A utoradiogram T e c h n iq u e
EXPERIMENTAL RESULTS ..............................................
21
E ffe c t o f GAP an d RIF on M a tu re P h ag e F o r m a tio n ....................
21
E ffe c t o f GAP on T4P o ly s o m e s .............................................................
21
E ffe c t o f CAP on T4 H ead C o a t P r o t e i n ................ 1.........................
25
DISCUSSION
. .................................................
54
SU M M A RY ...................................................................................................................
62
LITERATURE CITED
64
................................................. . . ....................................
vi
TABLE OF FIGURES
FIGURE
PAGE
1. C h lo ra m p h e n ic o l In h ib itio n C u r v e .................................................
28
2 . R ifam p icin In h ib itio n C u r v e ..............................................................
29
3 . C h lo ra m p h e n ic o l O ne S tep G row th C u r v e .................................
3 0 -3 1
4 . R ifam p icin O ne S tep G row th C u r v e ..............................................
32
5. T4D w . t . P o l y s o m e s ...........................................................................
3 3 -3 4
6. T4D w . t . P o ly so m e L y s is
C o n tro ls ............................................
35
7. T4D w . t . C h a s e d P o l y s o m e s ............................................................
3 6 -3 7
8 . T4D w . t . C h a s e d P o l y s o m e s ............................................................
38
9 . R ifa m p ic in T re a te d T4D w . t . P o ly s o m e s ....................................
39
10. P ro te in S y n th e s is A fter R ifam p icin an d
C h lo ra m p h e n ic o l T r e a t m e n t ............................. .............................
40
1 1. P ro te in S y n th e s is A fter R ifam p icin an d
C h lo ra m p h e n ic o l T r e a t m e n t .................................'.........................
41
1 2. P ro te in S y n th e s is A fter R ifam p icin and
C h lo ra m p h e n ic o l T r e a t m e n t ...........................................................
42
1 3. P ro te in S y n th e s is in T4amN54 P o ly s o m e s -..................................
4 3 -4 4
1 4. P ro te in S y n th e s is in T4am N 54 P o l y s o m e s ..................................
4 5 -4 6
1 5 . T4D P ro te in s in P o ly a c ry la m id e G e l s ................ .........................
4 7 -4 8
1 6. A u toradiogram o f T4D P2 3 H ead C o a t P r o t e i n ..........................
49
1 7 . A u to rad io g ram o f T4D P ro te in s R e s u ltin g from
R ifa m p ic in a n d C h lo ra m p h e n ic o l T r e a t m e n t ..........................
50
vii
1 8 . A utoradiogram o f R ifam p icin an d C h lo ra m p h e n ic o l
T re a te d T4D P r o t e i n s ........................................................................
51
1 9. P o ly a c ry la m id e G el F ra c tio n s S how ing C o m p le tio n
o f T4D P e p tid e s A fter C h lo ra m p h e n ic o l R e m o v a l................
52
2 0 . A utoradiogram S how ing C o m p le tio n o f T4amN54
P e p tid e s A fter C h lo ra m p h e n ic o l R em oval
.............................
53
viii
LIST OF TABLES
TABLE
I.
page
R ec o v e ry o f P ro te in S y n th e s is in C h lo ra m p h e n ic o l
In h ib ite d mRNA
57
ABSTRACT
In h ib itio n o f p ro te in s y n th e s is b y c h lo ra m p h e n ic o l (CAP) w as
c o m p le te ly r e v e r s ib le on b a c te rio p h a g e T4 mRNA, n a s c e n t p e p t i d e s ,
a n d h o s t E . c o li rib o s o m e s a f te r re m o v a l o f CAP. T h is w a s e s t a b ­
lis h e d by u s in g T4D w . t . an d T4 a m b er m u ta n ts amB17 a n d am N 54 w ith
d e n s ity g ra d ie n t p o ly so m e a n a ly s is a n d p o ly a c ry la m id e d is c g e l
e le c tro p h o re s is .
The mRNA c o n tin u e s to c o d e fo r p ro te in s y n th e s is a f te r tr a n s ie n t
tr e a tm e n t w ith CA P. T h is fin d in g w a s d e m o n s tra te d by f i r s t tr e a tin g T4
in f e c te d c e ll s w ith rifa m p ic in to p re v e n t fu rth e r mRNA s y n t h e s i s ,
w h ic h w a s fo llo w e d b y tre a tm e n t w ith CAP to in h ib it p ro te in s y n t h e s i s .
The CAP w a s s u b s e q u e n tly rem o v ed a n d th e u tiliz a tio n o f mRNA w h ic h
h a d b e e n form ed p rio r to rifa m p ic in tre a tm e n t w a s th e n t e s t e d b y o b ­
s e rv in g th e r a te o f C ^ a r g i n i n e in c o rp o ra tio n . T his in c o rp o ra tio n in ­
c r e a s e d s te a d ily d u rin g th e f i r s t 10 m in u te s a f te r CAP r e m o v a l, b u t 20
m in u te s l a t e r i t s r a te h a d d e c lin e d o n ly s lig h tly . T his r e s u l t in d ic a te d
th a t CAP tr e a te d mRNA re m a in e d fu n c tio n a l a n d c o n tin u e d to c o d e for
p ro te in s y n th e s is a f te r re m o v a l o f th e a n tib io tic .
M e s s e n g e r R N A -ribosom e c o m p le x e s w h ic h a re s u b je c te d to CAP
tre a tm e n t do re su m e p ro te in s y n th e s is a f te r rem o v al o f th e a n tib io tic .
N a s c e n t p e p tid e s la b e le d w ith C ^ - I e u c i n e w e re tra p p e d in th e p o ly ­
som e re g io n o f a s u c r o s e d e n s ity g r a d ie n t if CAP w a s a d d e d to T4 in ­
fe c te d c u l t u r e s . W ith th e s e c e ll s C ^ - I e u c i n e c o u ld b e c h a s e d o u t o f
th e p o ly so m e re g io n w h en C^2 - le u c in e w a s a d d e d a f te r CAP w a s r e ­
m o v e d . E ig h ty -fiv e p e r c e n t o f th is la b e l w a s rem o v ed from th is re g io n
in a 10 m in u te c h a s e in d ic a tin g th a t rib o s o m a l m ovem ent a n d fu n c tio n
h a v e b e e n r e s to r e d .
N a s c e n t p e p tid e s a re c o m p le te d an d form n o rm al h e a d c o a t p ro ­
te in a f te r tr a n s ie n t tre a tm e n t w ith CAP. T h is w a s d e m o n s tra te d on
p o ly a c ry la m id e g e ls by th e a p p e a ra n c e o f th e h e a d c o a t p ro te in band
w h en la b e le d n a s c e n t p e p tid e s in CAP tr e a te d c e lls w e re c o m p le te d
a f te r th e re m o v a l o f CAP to form n o rm al h e a d c o a t p ro te in .
T his w a s d e m o n s tra te d by a s e c o n d m ethod; in c o m p le te h e ad
c o a t p e p tid e s o f c e l l s tr a n s ie n t ly tr e a te d w ith CAP w e re c h a s e d in to
th e p ro te in a g g re g a te re g io n o f p o ly a c ry la m id e g e ls . T h e re fo re ,
n a s c e n t p e p tid e s in CAP tr e a te d c u ltu r e s a re c o m p le te d a n d r e le a s e d
n o rm a lly a f te r re m o v a l o f th e a n tib io tic .
INTRODUCTION
C h lo ra m p h e n ic o l
. E ver s in c e i t s d is c o v e r y in 1947, c h lo ra m p h e n ic o l (CAP) h a s
e lu d e d th e b io c h e m is t in h is a tte m p t to u n d e rs ta n d i t s m e c h a n ism o f
a c ti o n . S in c e c h lo ra m p h e n ic o l w a s fo u n d to in h ib it e f f e c tiv e ly p ro te in
s y n th e s is in b a c te r ia ( 1 ,2 ,3 ) , i t s com m on u s e in m o le c u la r b io lo g y a s a
b io c h e m ic a l to o l m ade i t d e s ir a b le th a t i t s a c tio n , b e u n d e rs to o d in
p r e c is e m o le c u la r t e r m s . The pro b lem h a s b e e n s tu d ie d im a g in a tiv e ly
by m any w o rk e rs .u s in g v a rio u s b a c t e r i a , p r in c ip a lly E s c h e r ic h ia c o l i .
D e s p ite th e s e e ffo rts i t s m e c h a n ism is r a th e r p o o rly u n d e r s to o d , p e r ­
h a p s b e c a u s e i t a c t s on th e c o m p le x '5OS rib o s o m a l s u b u n it o f b a c te r ia
( 4 , 5 , 6, 7 , 8 ) .
C h lo ra m p h e n ic o l d o e s n o t in te rfe re w ith am in o a c id a c tiv a tio n (9)
n o r w ith th e e s te r if ic a tio n o f tr a n s f e r rib o n u c le ic a c id w ith th e s e a c ­
tiv a te d am ino a c id s (10). It d o e s n o t in h ib it b in d in g o f am in o a c y ltRNA to rib o s o m e s (11,12,13,14) n o r th e b in d in g of m e s s e n g e r
r ib o n u c le ic a c id (mRNA) to rib o s o m e s (7,15,16,17,18).
14
In v itro s tu d ie s w ith C -CAP h a v e d e m o n s tra te d a w e a k b u t
s t e r e o s p e c if ic b in d in g to 70S rib o s o m e s (19,13). T h is b in d in g w as on
th e 508 rib o s o m a l p a r tic le a n d w a s r e a d ily r e v e r s ib le (4 ,2 0 ,1 3 ). A 1:1
e q u iv a le n c e b e tw e e n th e num ber o f rib o s o m e s an d th e n u m b er o f b o und
CAP m o le c u le s s u g g e s te d th e p r e s e n c e o f o n ly o n e b in d in g s ite for
2
CAP (13). O th e r w o rk e rs fin d th e 1:1 e q u iv a le n c e a t c o n c e n tra tio n s
b e lo w 50 [ig/m l CAP b u t sh o w b in d in g o f a p p ro x im a te ly tw o m o le c u le s
o f C A P /rib o so m e a t 200 n g /m l (5 ).
A n a ly s is o f ly s in e p e p tid e s from a CAP in h ib ite d p o ly a d e n y lic
a c id in v itro p ro te in s y n th e s is s y s te m in d ic a te d the. fo rm a tio n o f la r g e r
p e p tid e s (4-11 ly s in e r e s id u e s ) w a s in h ib ite d (21). T his w a s e x te n d e d
w h e n i t w a s sh o w n th a t n a s c e n t p e p tid e s d id n o t form on E . c o li p o ly ­
so m es, in th e p r e s e n c e o f CAP (5,1 7 ). T h e s e r e s u lts h a v e b e e n in te r ­
p re te d to m ean th a t CAP d ir e c tly b lo c k s som e ste p , in p e p tid e bond
fo r m a tio n .
The a n tib io tic p u ro m y c in , b e c a u s e o f its s tr u c tu r a l re s e m b la n c e
to a m in o a c y l-tR N A , e n te r s th e a m in o a c y l s ite o f rib o s o m e s an d b e ­
c o m es in c o r p o r a te d .in to th e grow in g p e p tid e c h a in . T h is c a u s e s th e
r e l e a s e o f th e p u ro m y c in -p o ly p e p tid e c h ain , from th e rib o s o m e . M any
w o rk e rs h a v e fo u n d CAP to in h ib it th is r e a c tio n (2 2 ,2 3 ,2 4 ,2 5 ,2 6 ) and
h a v e a rg u e d th a t CAP in h ib ite d th e rib o s o m a l p e p tid y l tr a n s f e r a s e r e ­
a c ti o n .
C a re fu l a n a ly s is o f th e .e ffe c t o f g u a n o s in e tr ip h o s p h a te (GTP)
in a s im ila r s y s te m c a u s e d W eb e r an d D e M o ss (27) to r e j e c t th e
p e p tid y l tr a n s f e r a s e r e a c tio n a s m e a s u re d b y th e p u ro m y cin . in d u c e d
r e l e a s e o f p e p tid e s from tRNA, b u t r a th e r in h ib ite d so m e s te p in th e
p e p tid e s y n th e s is c y c le p rio r to th is r e a c tio n .
W h en am ino a c id a u x o tro p h s o f E . c o li Rel (RC
) w e re s ta r v e d
for re q u ire d am in o a c id s RNA s y n th e s is s to p s a n d th e p o ly so m e re g io n
3
in s u c r o s e d e n s ity g ra d ie n ts w a s found, to be. re d u c e d (1.8) o r e n tire ly
-J-!
d e p le te d (16). The a d d itio n o f CAP a f te r s ta r v a tio n in Rel ■
' c e lls r e ­
g e n e r a te d norm al sed im en tin g , p o ly s o m e s in one m in u te , w h e re a s 8-10
m in u te s w ere, re q u ire d fo r an. e q u iv a le n t re g e n e ra tio n w h en th e re q u ire d
am in o a c id w a s a d d e d to s ta r v e d c e ll s in th e a b s e n c e o f CAP: (16).
G u rg o , A p irio n , a n d S c h le s s in g e r. (2 8) s u g g e s t th a t CAP u n ­
c o u p le s rib o s o m a l m o vem ent from p e p tid e bond fo rm atio n b e c a u s e - i t
a llo w s th e c o n tin u e d fo rm atio n o f p o ly s o m e s in th e a b s e n c e o f p ro te in
s y n t h e s i s . O th e r w o rk e rs h a v e d e m o n stra te d ..th a t C ^ - I a b e l e d n a s c e n t
p e p tid e s in itia te d in. th e a b s e n c e o f CAP w e re n o t c h a s e d .o f f th e s e
12
p o ly s o m e s d u rin g a 90 sec o n d , c h a s e w ith C -a m in o a c id s in. th e p r e s ’
--J-
e n c e o f CAP (17). F u rth e rm o re , CAP g e n e r a te d p o ly s o m e s in Rel
am ino a c id s ta r v e d c e ll s d id n o t c o n ta in n a s c e n t p e p tid e s . (16). The
r e s u lts o f W e b e r a n d D e M o ss a n d o f M o rris an d D e M o ss w e re c o n ­
firm ed -an d . e x te n d e d to show; th a t CAP p ro m o ted 50S a n d 308 rib o so m a l
s u b u n its in to p o ly s o m e s , an d th a t CAP g e n e ra te d p o ly s o m e s d id n o t
p o s s e s s n a s c e n t p e p tid e s 5 m in u te s a f te r th e rem o v al o f th e a n tib io tic
(18),. H o w e v e r, a f te r 25 m in u te s , la rg e am o u n ts o f n a s c e n t p e p tid e s
w e re p r e s e n t in th is p o ly so m e re g io n .
A n a ly s is o f n a s c e n t p e p tid e s on E . c o li p o ly s o m e s to. re s o lv e th e
a b o v e c o n tr o v e r s ie s h a v e e n c o u n te re d s e r io u s d i f f i c u l t i e s . T reatm en t
o f E . c o li w ith CAP a llo w e d c o n tin u e d RNA s y n th e s is a n d a n a c c u m u la ­
tio n o f CAP p a r tic le s (29) . T h e se p a r tic le s w ere c o m p le te d -and b e ca m e
4
f u n c tio n a l a f te r th e re m o v a l o f th e a n tib io tic ; T h erefo re , any/ la b e le d '
am ino a c id s a d d e d a f te r CAP re m o v a l c a n b eco m e in c o rp o ra te d in to
rib o s o m a l p ro te in s u s e d to c o m p lete; th e CAP .p a rtic le s . These: la b e le d rib o s o m a l p ro te in s w o u ld o b s c u re n a s c e n t p e p tid e p ro tein , s y n t h e s i s .
A lth o u g h w e ll d e f i n e d t h e . p o ly so m e re g io n o f E . c o ll la c k s a p red o m ­
in a n t s p e c ie s o f mRNA d u rin g g ro w th w h ic h w o u ld c o d e fo r a s in g le
p re d o m in a n t p r o t e i n . In g e n e ra l •, jE . c o li p o ly so m e s .h av e n o t b e e n u s e '■ 'v
ful to o ls in th e s tu d y o f th e m e c h a n ism o f actio n , o f CAP •
The u se. o f b a c te rio p h a g e . T4 c irc u m v e n ts some, o f th e s e d if f ic u l­
t i e s a n d w a s u sed . in. t h e s e s tu d ie s - to c o n trib u te -to . k n o w le d g e a b o u t
th e m e c h a n ism o f a c tio n o f CAP. S p e c if ic a lly e x a m in e d w ere- th e fa te
o f rib o js o in e s , mRNA, a n d n a s c e n t p e p tid e s a f te r tr a n s ie n t tre a tm e n t
w ith CAP.
R a tio n a le
W h en th e p h a g e T4 in f e c ts a h o s t b acteriu m , s e v e r a l c h a r a c te r ­
i s t i c e v e n ts o c c u r . A fter in je c tio n o f th e v ir a l .DNA, -no fu rth e r h o s t
DNA o r RNA s y n th e s is o c c u rs. (3 0 ,3 1 ). S y n th e s is o f h o s t p ro te in and
.ribo;somes. is a ls o sto p p e d , s h o rtly a f te r p h a g e in f e c tio n (3 2 ). Thus ,
• o n e is a llo w e d to t r e a t T4 in f e c te d E . c o li w ith CAP' a n d n o t in d u c e th e
fo rm atio n o f CAP p a r t i c l e s . The f a te o f rib o s o m e s , mRNA. a n d n a s c e n t
p e p tid e s c a n now. be. s tu d ie d by ..polysom e, a n a ly s is on s u c r o s e d e n s ity
g ra d ie n ts .
5
C e lls in f e c te d w ith b a c te rio p h a g e T4D p ro d u c e d p o ly s o m e s n e a r
th e e n d o f th e e c li p s e p e rio d in, th e p h a g e in tr a c e l lu la r lif e c y c le .
T h e se p o ly s o m e s w e re o b s e rv e d a s a p e a k m ig ra tin g in d e n s ity .g ra d i­
e n ts a t 2808 (3 3 ). C h y m o try p tic c le a v a g e .o f th e - is o la te d n a s c e n t p e p ­
tid e s fo u n d in th is p e ak , re v e a le d th a t th e m ajo r p ro d u c t w a s from
p h a g e g e n e 23 (3 4 ). The g e n e 23 p ro d u c t (P23) (35) w a s sh o w n to be
th e m a jo r h e a d c o a t p r o te in .o f m a tu re T4 (3 6 ).
I t w a s found th a t th e h e a d c o a t p ro te in o f T4 m ad e up. 85% o f th e
to ta l p h a g e p ro te in (3 7 ), an d th a t 60-70% o f p ro te in s y n th e s is o c c u r­
rin g la te in the-, p h a g e lif e c y c le w a s d e v o te d tO' p ro d u c tio n o f th is p ro ­
d u c t (38) . C o n s is te n t w ith th is w a s th e fin d in g th a t th e m a g n itu d e o f
th e 2 808 p o ly so m e p e a k in c r e a s e d w ith in c r e a s in g tim e, a f te r in fe c tio n
. u n til a m axim um w a s re a c h e d la te in in f e c tio n (3 4 ,3 9 );
I t w a s th e re fo re p o s s ib le to s tu d y a p o ly so m e, re g io n w h ic h p o s ­
s e s s e d , a w e ll d e fin e d peak, r e s p o n s ib le fo r th e s y n th e s is o f th e m a jo r
p h a g e p ro te in w h i c h ,i t s e l f w a s w e ll c h a r a c te r iz e d .
P h a g e p ro te in s , s p e c if ic a lly P23 , Were a s s a y e d b y d is c g e l
e le c tr o p h o r e s is . (40,41, 42,43) a n d a u to ra d io g ra p h y (40; 41). This
a llo w e d , ra p id h ig h r e s o lu tio n a n a ly s is o f phage, p ro te in s b y s ta in in g o r
b y th e ir in c o rp o ra te d r a d io a c tiv ity (4 '4 ). Amber m u ta n ts o f T4 (4 5 ,4 6 ,
47) w e re u s e d to e s t a b l i s h g e n e fu n c tio n w ith d is c r e te p ro te in s, on poly­
a c ry la m id e g e ls (4 0 ,4 1 ). The p ro te in P23 w a s found to b e th e m ajo r
6
, r a d io a c tiv e b a n d in th e s e g e ls d u rin g .la te , in fe c tio n and. w a s m o n ito red
w ith r e la tiv e e a s e .
In c o n c l u s i o n ,. th e u s e o f b a c te rio p h a g e T4 to stu d y ,-th e m e c h a n ­
ism o f a c tio n o f CAP e lim in a te d , m any p ro b le m s w h ic h d e v e lo p e d in .in
v itro E-. c p li s t u d i e s .
The a d v a n ta g e s - in c lu d e d : n o new. h o s t rib o so m e .
s y n th e s is a f te r in f e c tio n a n d th e re fo re no CAP p a r tic le s , a w e ll d e ­
fin e d p o ly so m e re g io n w h ic h s y n th e s iz e d p r in c ip a lly th e p h a g e h e a d
c o a t p r o te in , and. p o ly a c ry la m id e a n a ly s is o f th is c o a t p r o te in . T h e re ­
f o r e , i t was. p o s s ib le to d e term in e , th e f a te .o f mRNA, rib o s o m e s and
. n a s c e n t p e p tid e s a f te r tre a tm e n t an d re m o v a l o f CAP.
MATERIALS AND' METHODS
B a c te ria l an d B a c te rio p h a g e S tra in s
The b a c te r ia l s tr a in s u s e d w e re E s c h e r ic h ia c o ll B w ild ty p e
( w . t . ) , w h ic h i s a r e s tr ic tiv e h o s t fo r T4 am b er m u ta n ts , o b ta in e d
. from D r. P . D . S k a a r , M o n ta n a S ta te U n iv e r s ity , B o ze m a n , M o n tan a
and. E . c o ll CR63 , w h ic h is a s u p p r e s s o r h o s t fo r T4 a m b e r m u ta n ts ,
o b ta in e d from D r. G . S tr e i s in g e r , U n iv e r s ity o f O reg o n , E u g e n e ,
O re g o n .
The p h a g e s tr a in s u s e d w e re T4D w . t . an d a m b er m u ta n ts
T4a m B l7 a n d T 4am N 54. All w e re o b ta in e d from D r. G . S tr e i s in g e r ,
U n iv e r s ity o f O re g o n , E u g e n e O r e g o n .
M e d ia a n d S u p p le m e n ts
_A11 m e d ia a n d s u p p le m e n ts w e re s te r iliz e d in a n a u to c la v e fo r 20
m in u te s a t 15 p o u n d s p r e s s u r e u n le s s o th e r w is e - in d ic a te d . F in a l c o n ­
c e n tr a tio n s .are g iv e n in gram s (g) o r m illig ra m s ( m g ) /lite r (I) o f d i s ­
ti l l e d w a te r u n le s s o th e rw is e in d ic a te d .
M9 m in im al (M 9): N a^H P O ^,5.9 g; KH^PC^, 3.0 g; N H ^C l, 1.0 g;
M gSO ^-7 HgO, 0.25 g; g lu c o s e ,4.0 g; L -try p to p h a n , 20mg; FeClg, 2.7 m g.
14
All e x p e rim e n ts e m p lo y in g C - i s o t o p e s w ith T4 w e re c o n d u c te d in M9
m inim al: m e d ia .
8
M 9S: Sam e a s M9 m e d ia , e x c e p t th a t th e o tg a n ic co m p o u n d s .
g lu c o s e a n d L -try p to p h a n w e re .o m itte d . M9S w a s u s e d in s e r ia l d ilu ­
tio n s o f iT4 fo r d e te rm in in g , p h a g e t i t e r s .
N u trie n t b ro th (NB): B acto N u trie n t Broth, lO.g; N a C lz 5 g .
I
N u trie n t a g a r (NA): B acto N u trie n t Broth, 10 g; B acto -A g ar ,'.I^ g;
N a C l, S g .
Top la y e r a g a r (TL): B acto N u trie n t B roth,10 g; B ac to -A g a r,? g;
NaGl, S g .
C h lo ra m p h e n ic o l (CAP): T his w a s a g ift from P ark e D a y is C o .
an d w a s u s e d in 2 m g /m l s to c k s b lu tio n s .
This u n a u to c la v e d s o lu tio n
w a s s to re d a t 4 °C a n d re p la c e d a t l e a s t o n c e p e r m b n th .
R ifa m p ic in (RIF): T his is a m a c ro c y c lic a n tib io tic u s e d to in h ib it
DNA d e p e n d e n t RNA p o ly m e r a s e , (4 8 ,4 9 ). U n a u to c la v e d 2 m g /m l s to c k
s o lu tio n s w e re s to re d a t 4 °C in th e d a rk .
L -A rg in in e ( C ^ - a r g ) : 2 m g /m l s to c k s o lu tio n s u s e d in C ^ a rg in in e e x p e r im e n ts .
L -L e u c in e ( C ^ - I e u ) : 2 m g /m l s to c k s o lu tio n s u s e d in C ^ le u c in e e x p e r im e n ts .
U ra c il ( C ^ - u r a c il) : 2 m g /m l s to c k s o lu tio n s u s e d in C ^ - u r a c i l
e x p e r im e n ts .
9
B a c te ria l a n d B a c te rio p h a g e S to ck P re p a ra tio n
B a c te ria l s t r a i n s ; E . c o ll B a n d CR63 w ere grow n a n d s to re d a t
. 4 °C on NA s la n t s . C u ltu re s w e re tra n s fe rre d to n e w s la n t s o n c e p e r
m onth to e n s u r e ra p id g ro w th .o f th e o v e rn ig h t M9 m in im al c u ltu re s .
B a c te rio p h a g e s t r a i n s : P h ag e s to c k s o lu tio n s w e re o b ta in e d by
.grow ing E. c o li B to 5 XlO^ c e l l s / m l a t 3 7 °C and- in f e c tio n w ith T4D
w . t . a t a m u ltip lic ity , o f in fe c tio n (M OI) o f 5 . F iv e m in u te s a fte r i n ­
f e c tio n th e s e c e ll s w e re s u p e r in f e c te d w ith a m u ltip lic ity o f s u p e r in ­
fe c tio n . (MOSI) o f 5 . A e ra tio n a t 3 7 °C in .a N ew B ru n sw ick g y ra to ry
in c u b a to r s h a k e r w a s c o n tin u e d fo r 4 h o u rs w h en I m l/1 o f ch lo ro fo rm
w a s a d d e d . The s u s p e n s io n w a s th e n th o ro u g h ly m ix e d , a n d .a llo w e d
to s ta n d , fo r 15 m in u te s a t 4 ° C . The b a c te r ia l d e b ris from th is ly s a te
w a s re m o v e d by s p in n in g in s te r ile c e n trifu g e tu b e s fo r 15 m in u te s a t
SOOOrpm (4 0 8 0 Xg) in th e S o rv a ll c e n trifu g e GSA ro to r a t 4 ° C . T his
s u p e r n a ta n t w a s p la c e d , in to n e w s t e r i l e c e n trifu g e tu b e s, an d sp u n fo r
20 m in u te s a t SOOOrpm in .th e sa m e r o t o r , T his s u p e r n a ta n t w as c a r e ­
fu lly d e c a n te d a n d s to re d a t 4 °C o v e r ch lo ro fo rm an d h ad an. in itia l
t i t e r o f 2 -B XIO10 T 4 D /m l.
T h ese, p h a g e s to c k s o lu tio n s w e re c o n c e n tra te d fu rth e r w hen n e c ­
e ssa ry ,
b y p e lle tin g a t 12,000 rpm (21,500X g) for 60 m in u te s in th e
S o rv a ll GSA ro to r a t 4 ° C . The s u p e r n a ta n t w a s d e c a n te d an d th e p e l l e t
c o v e re d w ith 5 ml o f c o ld LB: b u ffe r a n d a llo w e d to d is s o lv e , for 12 h o u rs
in..the c o ld . The in s o lu b le d e b ris w a s rem o v ed b y a f in a l lo w s p e e d
10
s p in . The o p a le s c e n t s u p e r n a ta n t w a s - tite r e d an d d ilu te d to a w o rk in g
c o n c e n tra tio n , o f 2 - 4 X 1 0 ^ T 4 D /m l. This s to c k p re p a ra tio n m a in ta in e d
a s ta b le t i t e r for up. to 12 m o n th s .
The p re p a ra tio n o f T4 a m b e r m u ta n t s to c k s o lu tio n s w a s id e n tic a l
to th e p re c e d in g m e th o d e x c e p t th a t E . c o ll CR63 w a s used- in.-.place o f
E . c o li B an d th e s e c u ltu r e s w e re a llo w e d to grow fo r 8 h o u rs w ith
a e r a tio n a t 3 7 ° C .
B a c te rio p h a g e A s s a y
Phage, t i t e r s w e re d e te rm in e d , by th e a g a r o v e rla y m eth o d (50) .
S te rile , 10 cm p e tr i d is h e s w e re filld d w ith 20 ml o f NA. W hen th e s e
s o lid if ie d th e y w e re d rie d a t 3 7 °C fo r a t l e a s t 12 h o u rs . Two an d o n e h a lf ml o f s o f t 4 5 °C TL a g a r w a s in o c u la te d w ith 0,2 0 ml o f a 5 X 10®
c e l l s / m l c u ltu re o f Ev c o li B fo r -tite rin g T4D w , t . o r Ev c o li CR63 for
-tite rin g T4 a m b er m u ta n ts . P hag e s e r i a l d ilu tio n s w e re m ade w ith
0.1 ml o f th e a p p ro p ria te d ilu tio n a d d e d to th e TL a g a r c o n ta in in g th e
h o s t b a c t e r i a . T his TL a g a r w a s m ix ed and. poured, o n to th e NA. p l a t e s .
W hen, i t h a d h a r d e n e d , th e p la te w a s d e v e lo p e d a t 3 7 °C for 12 h o u rs .
G row th T e m p e ra tu re s
The p re p a ra tio n o f p h a g e s to c k s o lu tio n s , g ro w th of o v e rn ig h t c u l­
tu r e s
an d p h a g e titr a ti o n s w a s c a r r ie d .o u t a t 3 7 ° C . All o th e r stu cjies
w e re c o n d u c te d at- 3 0 °C in o rd e r to s lo w p h a g e i n tr a c e l lu la r d e v e lo p ­
m e n t, th u s a llo w in g m ore tim e to perform e x p e rim e n ta l p r o c e d u r e s . At
11
3 00 C , s p o n ta n e o u s h o s t l y s i s d id n o t b e g in u n til 40 m in a f te r in fe c tio n
. in M9 m inim al m e d ia .
O ne S tep G row th C u rv e s
T h e se w e re c a r rie d o u t a c c o rd in g to th e m eth o d o f E llis and
D e lb ru c k (51) , e x c e p t th a t th e c u ltu r e .s a m p le s w e re tr e a te d w ith ■
ch lo ro fo rm to ly s e th e b a c te r ia in w h ic h p h a g e e n d o ly s in w a s bfeing
form ed (5 2 ). E ach sa m p le w a s s e r i a l l y d ilu te d ; in M 9S a n d tite r e d by
th e a g a r o v e rla y m e th o d .
B io c h e m ic a ls , R e a g e n ts , an d S u p p lie s
H y d ro c h lo ric a c i d : R ea g e n t g ra d e ; E . I . D u p o n t C o m p an y .
G la c ia l a c e t i c a c id ; R e a g e n t g ra d e ; E . I . D u p o n t C o m pany.
T r ic h lo ro a c e tic a c id : A n a ly tic a l re a g e n t; M a llin c k ro d t C h e m ic a l
W o rk s .
S u c r o s e : M ann U ltra Pure; M ann R e s e a rc h L a b o ra to rie s , In c .
Y e a s t RNA: Y e a s t n u c le ic a c id ; M ann R e s e a rc h L a b o ra to rie s ,
Ihc.
PRO: 2 ,5 -D ip h e n .y la x o z a le ; P a c k a rd In s tru m e n t Co ., In c .
H y a m in e : H y d ro x id e o f h y a m in e 10X; P a c k ard In s tru m e n t C o .,
In c .
L y so z y m e : Egg w h ite , 3 X c r y s ta lliz e d ; Sigm a C h e m ic a l Com ­
pany .
12
T rizm a; T rizm a B a s e , re a g e n t g ra d e ; Sigm a C h e m ic a l C o m p an y .
D N a se I; D e o x y r ib o n u c le a s e , R N ase free, 215 0 u /m l; W o rth in g to n
B io c h e m ic a l C o r p o r a tio n .
R N ase B: R ib o n u c le a s e , c h ro m a to g r a p h ic a lly p u re ; W o rth in g to n
B io c h e m ic a l C o rp o ra tio n .
X -ra y film : 8 " X 1 0 " , NS54T; E a stm a n Kodak C o .
Kodak lig u id d e v e lo p e r ; E a stm a n Kodak C o .
K odak lig u id f ix e r ; E a stm a n K odak C o .
M illip o re f i l t e r s ; HAW P, n itr o c e llu lo s e ; M illip o re C o rp o ra tio n .
R a d io is o to p e s
The fo llo w in g C ^ - i s o t o p e s w e re o b ta in e d in 50 uC i lo ts from
A m e rs h a m -S e a rle C o r p o r a tio n . T hey w e re d ilu te d to a w o rk in g c o n c e n ­
tr a tio n .o f I iuC i/100 pi w ith g l a s s d i s t i l l e d w a te r an d s to r e d fro z e n
w h e n n o t in u s e .
L -A rg in in e-C 1 4 (U ) (C
■14
-a rg ): 312 m C i/m M .
L -L e u c in e -C 1 4 (U ) (C 14-Ie u ): 312 m C i/m M .
U r a c i l - 2 -14 ( C14-U ra c il): 52.5 m C i/m M .
B uffers
The fo llo w in g b u ffe rs w e re m ad e w ith g la s s d i s t i l l e d w a te r w h ic h
w a s h o u s e d i s t i l l e d w a te r r e d i s t i l l e d in a w a te r d i s t i l l a t i o n a p p a r a tu s .
13
The pH o f th e s e b u ffe rs w a s a d ju s te d u s in g g l a s s d is tille d
h y d ro c h lo ric a c id w h ic h w a s th e m id d le th ird d i s t i l l a t e o f a 1:1 d ilu ­
tio n w ith g l a s s d i s t i l l e d w a te r o f r e a g e n t g ra d e h y d ro c h lo ric a c id .
T h e s e b u ffe rs w e re tr e a te d w ith 2 d ro p s / I o f a b e n to n ite s u s p e n ­
s io n fo r 2 h o u r s . T his c o llo id w a s know n to b in d R N ase a n d w as
th e re fo re u s e d to rem o v e th is e n zy m e from th e b u ffe rs .
B; T rizm a b a s e , 12.11 g; K C l, 3.72 g; Mg(OAc) ^ , 2.359 g . T h e se
c r y s ta ll in e co m p o u n d s w ere d is s o lv e d in I lite r - w a te r a n d th e pH
a d ju s te d to 7.8 a t 4 ° C . A fter tre a tm e n t w ith b e n to n ite , th is b u ffe r w a s
f ilte r e d w ith HAWP M illip o re f ilte r s in to h e a t tr e a te d re a g e n t b o ttle s
a n d s to re d a t 4 ° C .
LB: T rizm a b a s e , 12.11 g; N a C l , 5.84 g; Mg(OAc) ^ , 2.16 g . T his
b u ffe r w a s tr e a te d a n d s to re d th e sa m e a s B uffer B a b o v e .
R ib o n u c le a s e R em oval
R ib o n u c le a s e (R N ase) w a s rem o v ed from e x p e rim e n ta l h a rd w are
b y tw o m e th o d s: h e a tin g for 2 h o u rs a t 210°C in a h o t a ir o v e n or a u to ­
c la v in g fo r 15 m in u te s a t IS p o u n d s p r e s s u r e . R N ase w a s rem o v ed from
b u ffe rs b y a d d in g 2 d ro p s /1 o f a b e n to n ite s u s p e n s io n w h ic h w as r e ­
m oved b y f iltr a tio n th ro u g h M illip o re f ilte r s .
T h is p ro c e d u re w a s p a r tic u la r ly im p o rta n t in p o ly s o m e a n a ly s is
b e c a u s e a n y r e s id u a l R N ase on g la s s w a r e o r in b u ffe rs c o n trib u te d to
th e 70S rib o s o m e re g io n s by d e g ra d a tio n of m R N A -ribosom e c o m p le x e s .
14
G e n e ra l E x p e rim e n ta l P ro ce d u re
An o v e rn ig h t c u ltu re o f E . c o li B w a s d ilu te d 1:10 w ith p re ­
w arm ed M9 and. a e r a te d g e n tly a t 3 0 °C in a N ew B ru n sw ick m e ta b o ly te
w a te r b a th s h a k e r . The o p tic a l d e n s ity (O. D .) o f th is c u ltu re w as
m e a s u re d p e r io d ic a lly w ith a B au sc h a n d Lomb S p e c tro n ic 20 s p e c tr o ­
p h o to m e te r a t 52 5 mp. u n til 0.2 0 0 . D. w a s re a c h e d (a p p ro x im a te ly
'2 X lO 8 c e l l s / m l ) .
T h e se c e ll s w e re in f e c te d a t O .D. = 0.2 0 w ith T4D w . t . o r T4
a m b er m u ta n ts a t a n M QI = 5 an d a f te r 5 m in u te s s u p e r in f e c te d vi/if'h a n
MOSI== 5. T his c u ltu re w a s d iv id e d in to 10. o r 20 ml a liq u o ts a h d z
a e r a te d a t 3 0 °C u n til 15 o r 20 m in u te s a f te r in f e c tio n .
C arb o n ^ -
is o to p e s w e re a d d e d a t th e s e tim e s s in c e th e s y n th e s is o f h e a d c o a t
p ro te in (P23) w as- o c c u rrin g a t a n e a r m ax im al ra te and. 2 0 m in u te s s t i l l
re m a in e d in th e p h a g e in tr a c e l lu la r lif e c y c le b e fo re s p o n ta n e o u s ly s i s
b e g a n . O n e - h a lf o r o n e .M-Ci a liq u o ts , o f C ^ - a r g in in e o r C ^ - I e u c in e
la b e le d th e s e , in f e c te d c e lls a d e q u a te ly d u rin g a o n e m inute, p u ls e .
T his p ro d u c e d a w e ll d e fin e d p o ly so m e re g io n .
The a n tib io tic s RIF and CAP w e re u s e d a t v a ry in g tim e s a fte r i n ­
fe c tio n , b u t a lw a y s a t c o n c e n tra tio n s w h e re mRNA or p ro te in s y n th e s is
w e re c o m p le te ly in h ib ite d . W h e n e v e r C ^ - is o to p e s o r CAP w ere r e ­
m oved from th e s e c e ll s one o f tw o m e th o d s w as u s e d : a 1:100 d ilu tio n
o f c e ll s w ith p rew arm ed M9 o r b y p e lle tin g th e c e ll s a t BOOOrpm for 5
m in u te s in th e S o rv a ll S S -I r o t o r . T h e se c e lls w e re c h ille d - b e fo re
15
c e n trifu g a tio n , to. 0 °C w ith o n e - h a lf v o lu m e o f ic e an d w ere r e s u s p e n d ­
ed in prew arm ed. M9. to c o n tin u e th e e x p e rim e n t.
C a rb o n i4- a r g in in e , ‘C ^ - l e u c i n e , an d C ^ - u ra c il w e re e f f e c tiv e ly
c h a s e d w ith 50 [ig/m l o f th e u n la b e le d c o rre s p o n d in g c o m p o u n d . C e ll
g ro w th w a s s to p p e d by p o u rin g th e c e ll s o v e r o n e - h a lf v o lu m e o f ic e
an d p e lle tin g them , in th e S o rv a ll c e n tr if u g e .
C e ll L y s is P ro ce d u re
411 ste p s, in. th is ly s i s p ro c e d u re w ere c a rrie d , o u t a t 0 -4 ° C u n ­
l e s s o th e rw is e in d ic a te d .
The s u p e r n a ta n t from p e lle te d c e ll s w a s p o u re d o ff an d a n y r e ­
m a in in g d ro p le ts w ip e d from th e u p p e r h a lf o f th e c e n trifu g e tu b e s .
The: re m a in in g c e ll p e l l e t w a s re s u s p e n d e d w ith 0.7 ml c o ld LB; b u ffe r
an d th e n q u a n tita tiv e ly tra n s fe rre d tO' h e a t tr e a te d 12 ml c o n ic a l c e n ­
trifu g e tu b e s .
Egg w h ite ly s o z y m e (1.2 mg) w a s a d d e d in 0.3 ml o f LB> b u ffe r
along, w ith 4 |J.g o f d e o x y rib o n u c le a s e (D N a se I ) . T h e s e c e ll s w ere
fro zen , in. a - 9 0 ° C a lc o h o l b a t h , th aw ed , fo r 30 m in u te s in a n ic e - w a te r
b a th , h e a te d to 30OC. for 2 m in u te s a n d th e n c h ille d .to 0 ° C . This
f r e e z e - th a w .tr e a tm e n t w a s r e p e a t e d . T h e s e -ly s e d c e l l s w ere im m ed i­
a t e l y la y e r e d on s u c r o s e d e n s ity g r a d ie n ts w ith h e a t tr e a te d I ml s e r o ­
lo g ic a l p i p e t t e s , or m ade SM w ith r e s p e c t .to u re a b y a d d in g 0.48 g o f
c r y s ta ll in e u re a to th e c e ll ly s a t e a s th e e x p erim en t, r e q u ir e d .
16
T his l y s i s p ro c e d u re w a s u s e d fo r a ll T4D s tu d ie s e x c e p t fo r
a m b er m u ta n t T4am N 54 w h ic h w a s ly s e d b y th re e c y c le s o f f r e e z e ­
th a w in g w ith o u t h e a tin g to 30OC .for 2 m in u te s . T h e se ly s e d c e lls w ere
s p u n a t 4°C . for 30 m in u tes, a t h ig h s p e e d in th e #221 s w in g in g b u c k e t
h ead, o f th e I n te r n a tio n a l c lin ic a l c e n tr if u g e . T his s u p e r n a ta n t w a s
th e n c a r e f u l ly la y e r e d on 15-30% s u c r o s e g r a d ie n ts .
R ib o n u c le a s e T re a tm e n t
A c l a s s i c a l m ethod, in c h a ra c te riz in g .-p o ly s o m e m a te ria l :
fo u n d s e d im e n tin g f a s te r - th a n 70S rib o so m e m onom ers in a s u c ro s e
d e n s it y g r a d i e n t , is to d e m o n s tra te th e ir s e n s itiv ity to lo w le v e ls o f
R N ase (5 3 ). If th is f a s t s e d im e n tin g m a te ria l re p re s e n ts , p o ly so m e s i t
is d e g ra d e d an d fo u n d a s a la rg e p e a k in th e 70S re g io n o f a d e n s ity
g r a d ie n t. The c e ll ly s a t e in th e s e s tu d ie s w as tr e a te d w ith 2 |ig /m l
R N ase B for 10 m in u te s a t 3 0 ° C . T h is ly s a te w as .im m e d ia te ly c h ille d
to 0 °C and. la y e r e d on s u c r o s e g r a d ie n ts .
SucfosO D e n s ity G ra d ie n t P ro ce d u re
P re p a ra tio n : S u c ro s e s o lu tio n s o f 15, 30 a n d .50%' (w /w ) w ere
m ade by a d d in g 150, 300 an d .500 g r e s p e c tiv e ly o f s u c r o s e to m ake o n e
k ilo g ra m s o lu tio n s . T h e se w ere s to re d in s te r ile r e a g e n t b o ttle s in th e
4 C c o ld . room, w h e re th e y w e re form ed in to l i n e a r g r a d ie n ts . The .gra)d ie n ts w e re c o n s tr u c te d by a d d in g 0.5 ml 50% s u c r o s e to th e bottom, o f
S p in c o s w 2,5.3 ro to r c e llu lo s e n itr a te c e n trifu g e t u b e s , th e n la y e rin g a
17
lin e a r 13 m l, 15 to 30% (w /w ) s u c r o s e g ra d ie n t in to th e s e tu b e s . The
g ra d ie n ts w e re made, up a t l e a s t s ix h o u rs p rio r to c e n tr ifu g a tio n so
th a t s lig h t d e n s ity ir r e g u la r itie s c o u ld e q u ilib r a te .
■ C e n trifu g a tio n : O ne ml o f c e ll ly s a t e w a s c a r e f u lly la y e re d o n to
th e s e g ra d ie n ts w ith h e a t - t r e a t e d I ml s e r o lo g ic a l p i p e t t e s . They w ere
sp u n 2.25 h o u rs a t 25,000 rpm in th e S p in c o L-2 u ltr a c e n trifu g e sw 2 5 .3
ro to r . At th e c o n c lu s io n o f th e run th e ro to r w as a llo w e d to sto p w ith
th e c e n trifu g e b ra k e in th e OFF p o s i t i o n . The c e n trifu g e b u c k e ts w e re
rem o v ed from th e ro to r an d s to re d in a re frig e ra to r u n til f r a c tio n a tio n .
F r a c tio n a tio n : S ix te e n drop fra c tio n s , o f th e s u c r o s e d e n s ity
g r a d ie n t w e re c o lle c te d in- h e a t - t r e a t e d 12 X 150 mm tu b e s w ith 'a d ro p ­
c o u n tin g fr a c tio n c o lle c to r by p u n c tu rin g th e bottom o f th e g ra d ie n t
t u b e . D uring fr a c tio n a tio n th e g ra d ie n t w a s h e ld in a 4 °C c o o lin g
ja c k e t.
P r e c ip ita tio n a n d P la tin g P ro c e d u re fo r D e n s ity G ra d ie n t F ra c tio n s
C a rrie r y e a s t RNA (I m g /g r a d ie n t fra c tio n ) w a s ad d ed , to e a c h
f r a c tio n , th o ro u g h ly m ix e d , an d c h ille d to O0 C . T h re e -fo u rth s ml o f
c o ld 10% tr ic h lo r o a c e tic a c id (TCA) w a s a d d e d to e a c h f r a c tio n , m ix e d ,
an d th e r e s u ltin g p r e c ip ita te a llo w e d to f lo c u la te a t 4 ° C fo r a t l e a s t
30 m in u te s .
The p r e c ip ita te from e a c h fr a c tio n w as c o lle c te d on M illip o re
f il te r s u s in g a 30 sa m p le M illip o re m a n ifo ld . T h e se f il te r s w ere
18
w a sh e d , tw ic e w ith 2 -3 ml o f 5% TCA in. o rd e r to w a s h .o u t a ll u n p re c ip i­
ta te d . r a d io a c tiv ity .
T h e se .f ilte r s w e re m o u n ted o n c o p p e r d is c s w h ic h w ere. p la c e d
•:in d is c h o l d e r s , dried, fo r 10 m in u te s a t 1 6 0 °C , a llo w e d to c o o l to room
te m p e ra tu re an d counted, in. a N u c le a r C h ic ag o ' lo w - b a c k g ro u n d p la n c h e tte c o u n te r.
A c id ic P o ly a c ry la m id e U rea D is c G e l E le c tro p h o re s is
T h e s e p ro c e d u re s w e re c a r rie d o u t a c c o rd in g to th e m eth o d s o f
D a v i s .(42) w ith the. fo llo w in g m o d if ic a tio n s . A c id ic p o ly a c ry la m id e
g e l p ro c e d u re s a n d s o lu tio n s w e re a d o p te d from R eis fie ld . (54) a n d .th e
in c o rp o ra tio n o f SM u re a in to th e s e - g e ls from H o so d a an d L e v in th a l
. (4 0 ). A fter s e p a r a tio n .o f th e T4 p ro te in s , fo llo w e d b y s ta in in g an d d e s t a i n i n g ,. th e s e g e ls w e re s lic e d , lo n g itu d in a lly an d d rie d b y th e m e th ■o d s o f F a irb a n k s (4 4 ).
L iq u id S c in tilla tio n C o u n tin g o f P o ly a c ry la m id e G e ls
I
I
The s e p a r a tio n g e l p o rtio n o f p o ly a c ry la m id e g e ls c o n ta in in g ' '
.
C "^ -iso to p e ..to b e - a s s a y e d b y ,liq u id s c in t illa tio n c o u n tin g w ere s lic e d
. in to 0.5 cm. le n g th s , im m e d ia te ly a t th e c o n c lu s io n , o f e le c tr o p h o r e s is
- w ith o u t fix in g in. 7% a c e t i c a c id o r sta in in g , th e p r o te in . E ach fr a c tio n
w a s p la c e d in a 12 X150 mm P y rex tu b e and. fin e ly g ro u n d w ith a h o m o g e n iz e r . Otie m illi lite r o f hy am in e p ro te in s o lu b iliz e r w a s a d d e d to
e a c h fr a c tio n an d a llo w e d to s ta n d , fo r .12 hours, to e x tr a c t a ll p o s s ib le
19
p r o t e i n . T h e se h y a m in e -Solutions w e re p la c e d in ,v ia ls w ith 10 ml o f
s c in tilla tio n , flu id c o n ta in in g -507'ml to lu e n e , 415 ml a b s o lu te e t h a n o l ,
77 ml form am ide , and. 2.9 g P P O /lite r . T h e se s a m p le s w e re c o u n te d in
• a N u c le a r C h ic a g o 1liq u id s c in t illa tio n s p e c tro m e te r.
. A u toradiogram T e c h n iq u e
• 14
A u to ra d io g ra m s o f C . -T 4 p ro te in s w e re m ade b y d ry in g the- p o ly ­
a c ry la m id e g e ls a c c o rd in g to th e m e th o d s o f F a irb a n k s (44) and. p la c in g
t h e s e - f l a t , d rie d g e l s l i c e s i n X -ra y film c o n ta in e r s .
The X -ra y .film
w a s e x p o s e d to th e C 14- I s o to p e in..the d rie d g e ls fo r v a ry in g le n g th s o f
-tim e , b u t com m only fo r 24 h o u rs w h en I |_iCi C 14- I e u c in e w a s p u lsed , for
I m in u te in to 20 ml o f T4 in f e c te d E . c p li B- a t 3 0 ° C .
T h e s e ,film s w e re d e v e lo p e d w ith. Kodak liq u id d e v e lo p e r fo r 4
m in u te s and. d e v e lo p m e n t sto p p ed , w ith d ilu te a c e t i c a c id tre a tm e n t fo r
I m in u te . The film s w ere fix e d w ith Kodak liq u id fix e r fo r 10 m in u te s ,
■soaked in d i s t i l l e d w a te r for 30 m in u te s , and. a ir d r i e d .
T erm inology
P o ly so m e: In -this m a n u s c rip t th e te rm .p o ly so m e d.s a p p lie d to ma­
t e r i a l s e d im e n tin g f a s t e r than. 70S -rib o so m a l m onomers- in s u c ro s e
d e n s ity g r a d i e n t s . It w a s c o n s id e re d to c o n ta in .rib o s o m e s h e ld to ­
g e th e r by mRNA (5 5 ,5 6 ,5 7 ) w h ic h c a n be d eg rad ed , w ith , p a n c r e a tic
I
. f ib o n u c le a s e .(R N ase) form ing fre e 70S rib o s o m e s b u t n o t d e g ra d e d w ith
p r o te in a s e s o r D N a se (5 3 ). The s i t e o f c e llu la r p ro te in , s y n th e s is w a s
20
■ ]A
c o n s id e re d ; to be-the- p o ly so m e b e c a u s e C -a m in o a c id s could be
12
• p u ls e d in to i t a n d th e n c h a s e d o u t w ith C -a m in o a c id s d u rin g p ro te in
s y n th e s is .
Amber m u ta n ts : T h e se a re m u ta tio n s in. T4 w h ic h c a u s e p o ly p e p ­
tid e c h a in te rm in a tio n p re m a tu re ly .
T hey a re th e r e s u l t o f a b a s e
m u ta tio n w h ic h c o n v e r ts a n am ino a c id s p e c ify in g codon, in to th e b a s e
.. s e q u e n c e UA G , w h ic h s ig n a ls c h a in te r m in a tio n .
EXPERIMENTAL RESULTS
E ffe c t o f CAP a n d RIF on M atu re P h ag e F o rm atio n
The c o n tin u e d g ro w th o f b a c te r ia a f te r a b rie f tr e a tm e n t w ith CAP
h a s b e e n e x te n s iv e ly s tu d ie d (5 8 ,5 9 ,6 0 ) b u t l i t t l e is know n a b o u t th e
r e v e r s i b ility o f p r o c e s s e s in p h a g e in f e c te d b a c t e r i a . The f i r s t s te p in
th e p r e s e n t s tu d ie s e x a m in e s th e s e n s i t i v i t i t y o f th e p h a g e g row th to
CAP a n d RIF an d th e k in e tic s o f p h a g e fo rm atio n a f te r tr a n s i e n t a n t i ­
b io tic tr e a tm e n t.
The d a ta in d ic a te d in F i g s . I a n d 2 sh o w th a t fo rm atio n , o f m atu re
p h a g e w a s c o m p le te ly in h ib ite d b y 8 d g /rn l CAP o r 40 U g /ml R IF . T h e se
in h ib itio n c u rv e s w e re u s e d to s e l e c t a n tib io tic c o n c e n tr a tio n s w h ere
th e b u lk o f p ro te in s y n th e s is an d v ir tu a lly a ll th e m a tu re p h a g e fo rm a­
tio n w e re i n h i b i t e d .
The e f f e c t o f 50 u g /m l CAP on p h a g e p ro d u c tio n w a s c o m p le te ly
r e v e r s ib le (F ig . 3 ). By c o n t r a s t , th e e f f e c t o f 50 iag/m l RIF on p h a g e
p ro d u c tio n w a s ir r e v e r s ib le (F ig . 4 ). N o re c o v e ry in p h a g e p ro d u c tio n
w a s fo u n d w h e n RIF tr e a te d c e ll s w e re d ilu te d 1:100 in to f r e s h p re ­
w arm ed M9 m e d ia , o r w hen. RIF w a s r e ta in e d in th e c u ltu r e s th ro u g h o u t .
th e e x p e rim e n t.
E ffe c t o f CAP on T4 P o ly so m e s
In f e re n c e s re g a rd in g th e e f f e c t o f CAP on p o ly s o m e s an d th e ir
c o n s titu e n ts ( r ib o s o m e s , mRNA, an d n a s c e n t p e p tid e s ) c o u ld n o t b e
22
m ade from th e p re c e d in g e x p e rim e n ts . By u s in g th e T4 s y s te m , th e
fo llo w in g q u e s tio n s c o u ld be p o s e d ; is mRNA a llo w e d to co d e for fu r­
th e r p ro te in a f te r CAP in h ib itio n ; do tr a n s ie n tly in h ib ite d rib o so m e s
c o n tin u e to s y n th e s iz e p ro te in ; do n a s c e n t p e p tid e s c o m p le te n o rm a lly
a f te r re m o v a l o f CAP ?
R ap id ly s e d im e n tin g p o ly s o m e s w d re found in 15-30% s u c ro s e
g ra d ie n ts w h en T4D in f e c te d E . c o li B w ere ly s e d n e a r th e end o f th e
e c li p s e p e rio d (3 3 ,3 4 ). T his po ly so m e, re g io n c o n ta in e d a r e la tiv e ly
h o m o g e n o u s , w e ll d efin ed , p e a k a t g ra d ie n t f r a c tio n s 10-15 a s d e te c te d
by C ^ - I a b e le d n a s c e n t p e p tid e s (F ig s . 5 , 1 3 ) or C ^ - I a b e le d mRNA
(F ig . 9 ). T his p o ly so m e re g io n w a s e x tre m e ly la b ile to lo w le v e ls of
R N ase B an d w a s m oved from g ra d ie n t fr a c tio n s 10-14 to fra c tio n s
21-22 w h ic h r e p r e s e n te d fre e 70S rib o s o m e s ( F ig s . 5, 7, 9, 14). The
p r e s e n c e o f 70S rib o s o m e s in fr a c tio n s 21-22 w as v e r if ie d b y s e d i ­
m e n tin g p u rifie d 70S p re p a ra tio n s in to 15-30% s u c r o s e d e n s ity
g ra d ie n ts .
Q u a n tita tiv e d iffe re n c e s, in re fe re n c e p o ly so m e p e a k s r e s u lte d
from v a rie d ly s i s p ro c e d u re s w ith eg g w h ite ly so z y m e (F ig . 6). Two
c y c le s o f f r e e z e - th a w ly s is r e s u lte d in a smafll p o ly so m e p e a k , w h ile
tw o c y c le s o f f r e e z e - t h a w ly s is w ith tw o m in u te s o f h e a t a t 3 0 °C d r a ­
m a tic a lly in c r e a s e d th is p e a k . H o w e v d r, w h en th is h e a tin g w a s in ­
c r e a s e d to fo u r m in u te s , d e g ra d a tio n o f p o ly so m e s a n d th e a p p e a ra n c e
o f a 70S p e a k o c c u r r e d .
23
W h en Ci4- a r g in in e w a s p u ls e d in to T4D w . t . p o ly so m e p e a k s
(F ig . 7) , th e n c h a s e d fo r 10 m in u te s w ith 10 t-ig/ml C - a r g i n i n e , an u n u n e x p e c te d r e s u lt w a s fo u n d . I n s te a d o f b e in g r e a d ily re m o v e d , th e
14
C - l e v e l in .th e p e a k w a s in c r e a s e d d r a m a tic a lly , a s c o m p ared to th e
14
I m in u te C - a r g in in e p u l s e . The u n c h a s e d p o ly so m e p e a k w a s com ­
p le te ly s e n s itiv e to R N ase (F ig . 7 ), w h ile th e .ch ased , p e a k w as e n ­
tir e ly R N ase r e s i s t a n t (F ig . 8 ). Thus , T4D. p o ly so m e C ^ - n a s c e n t
p e p tid e s w e re c h a s e d in to a n R N ase r e s i s t a n t p e a k w h ic h w a s s u p e r ­
im p o s e d on th e R N ase s e n s itiv e p o ly so m e p e a k in th e d e n s ity
g r a d ie n ts ( F ig . 8) .
14
O n ly a s m a ll am o u n t o f C - u r a c i l w a s in c o rp o ra te d in to mRNA in
th e p r e s e n c e o f R IF . T his w a s d e m o n s tra te d w ith a c u ltu re c o n ta in in g
100 p g /m l RIF w ith a 5 m in u te C" ^ -u ra c il p u ls e (F ig . 9) . The C" ^ -u ra c il
in c o rp o ra te d in to th is p o ly so m e re g io n b o th in th e a b s e n c e an d p r e s ­
e n c e o f RIF w a s e n tir e ly R N ase s e n s i t i v e . S in c e RIF e f f e c tiv e ly
s to p p e d s y n th e s is o f mRNA, i t w a s u s e d to e x am in e p ro te in s y n th e s is
d ir e c te d by mRNA m ade p re v io u s to th e tr a n s ie n t tre a tm e n t w ith CAP in
o rd e r to a n s w e r th e fo llo w in g q u e s tio n . Is mRNA a llo w e d to co d e fo r
fu rth e r p ro te in s y n th e s is a f te r re m o v a l o f CAP?
14
W h en C - a r g in in e w a s a d d e d a t I, 10, and 20 m in u te s a fte r th e
1 14
re m o v a l o f RIF from c u ltu r e s , a s te a d y lo s s in C - in c o rp o r a tio n w ith
in c r e a s in g tim e w a s o b s e rv e d (F ig s . 10, 11, 12). A fter 20 m in u te s , no
C ^ - a r g in in e w a s found in .th e p o ly so m e re g io n . W h en c u ltu re s w ere
24
tr e a te d w ith CAP an d RIF th e la b e le d p o ly so m e p a tte r n w a s v e ry d if f e r ­
e n t ( F i g s . 10, 11, 12). In c o rp o ra tio n o f is o to p e in t h e s e p o ly so m e r e ­
g io n s .( F ig s . 10, 11, 12) in c r e a s e d fo r 10 m in u te s a f te r re m o v a l o f th e
d ru g s . W h ile a s lig h t drop w a s n o te d a t 2 0 m in u te s (from 63% to 54%) ,
a la rg e a m o u n t o f a c ti v ity re m a in e d . A sum m ary o f d a ta from th is e x ­
p e rim e n t is fo u n d in T ab le I o f th e d i s c u s s i o n .
The in f e c tio n o f E . c o ll B w ith T4am N 54 p ro d u c e d a p o ly so m e
re g io n w ith d i s t i n c t d if f e r e n c e s from T4D w . t . ( F ig ..13). A s e p a r a te
s p e c ie s o f p o ly s o m e w a s s e e n a t g r a d ie n t fra c tio n 4 a lo n g w ith th e
14
m a jo r p o ly s o m e p e a k a t f r a c tio n s 10-14. W h en th is C - I e c u in e la b e le d
p o ly s o m e re g io n w a s c h a s e d for 10 m in u te s a t 3 0 °C w ith 50 p g /m l
12
C - l e u c i n e , v ir tu a lly a ll o f th e r a d io a c tiv ity w a s m oved in to th e
s u p e r n a ta n t re g io n o f th e g r a d ie n t (F ig . 14). T h is p o ly s o m e re g io n w a s
a l s o c o m p le te ly s e n s i t i v e to R N a s e .
P o ly so m e a n a ly s is o f T4am N 54 in f e c te d c u ltu r e s a llo w e d e x a m i­
n a tio n o f th e fo llo w in g q u e s tio n : do CAP in h ib ite d rib o s o m e s in mRNArib o s o m e c o m p le x e s c o n tin u e to s y n th e s iz e p ro te in a f te r re m o v a l o f
CAP?
T h e s e in f e c te d c u ltu r e s w e re t r a n s ie n t ly tr e a te d w ith 50 p g /m l
12
CAP a n d th e n c h a s e d w ith 50 u g /m l C - le u c in e w ith th e fo llo w in g r e ­
s u l t s . A fter a 10 m in u te c h a s e 85% o f th e C ^ - I e u c in e w a s c h a s e d o u t
o f th e CAP tr e a te d p o ly so m e re g io n (F ig . 13). T his w a s in c r e a s e d to
9 7% a f te r a 2 0 m in u te c h a s e .
25
The r e s u l t s from th e RIF t r e a t e d c u ltu r e s i n d i c a t e d th a t mRNA
m ad e p rio r to CAP tr e a tm e n t c a n b e u s e d in c o d in g fo r p ro te in s y n t h e ­
s i s a f t e r re m o v a l o f CAP. The fin d in g t h a t n a s c e n t p e p t i d e s are
c h a s e d o u t o f th e T4amN54 p o ly s o m e re g io n w h en CAP w a s rem o v ed
i n d i c a t e s t h a t r ib o s o m a l m o v e m e n t c o n tin u e s , a n d im p lie s , b u t d o e s
n o t p r o v e , t h a t th e n a s c e n t p e p t i d e s a re r e l e a s e d n o rm a lly .
E ffe c t o f CAP on T4 H e a d C o a t P ro te in
The p r e c e d in g e x p e r im e n ts d id n o t p ro v id e c o n v in c i n g e v id e n c e
t h a t t r a n s i e n t l y i n h i b i t e d n a s c e n t p e p t i d e s form ed c o m p le te d p h a g e
p r o te in a f t e r re m o v a l o f CAP. The a n a l y s i s o f T4 p r o te in s on p o ly ­
a c r y la m id e g e l e l e c t r o p h o r e s i s w a s u n d e rta k e n to a n s w e r t h i s '
q u e stio n .
The p r o te in s o f T4 h a v e b e e n s e p a r a t e d a n d m a n y h a v e b e en
i d e n t i f i e d a s to g e n e fu n c tio n b y s u b j e c t i n g T4 a m b er a n d te m p e ra tu re
s e n s i t i v e m u ta n t l y s a t e s to p o ly a c r y la m id e g e l e l e c t r o p h o r e s i s (40,41,
3 5 ). S e p a r a tio n o f T4 p h a g e p r o te in s by a c i d i c u re a p o ly a c r y la m id e
g e l s w a s sh o w n (F ig . 15). This d ia g ra m d i s p l a y s d u p l i c a t e g e l s ,
s t a i n e d w ith a m id o s c h w a r z , th e d rie d s e c t i o n s o f t h e s e g e l s , and
a u to ra d io g ra m s o f th e s a m e d rie d g e l s .
G el a u to ra d io g ra m "c" s h o w s
th e i n t e n s e r a d i o a c t i v e b a n d m a rk e d P2 3 w h ic h w a s c h a s e d ou t o f g e l
12
a u to ra d io g ra m "f" w ith C - l e u c i n e .
The h e a d c o a t p r o te in P2 3 w a s id e n t i f i e d by in tr o d u c in g C ^ l e u c i n e a t i n c r e a s i n g tim e s a f t e r i n f e c t i o n a n d o b s e r v in g th e
26
a p p e a r a n c e o f an i n t e n s e r a d i o a c t i v e 'b a n d (F ig . 16). The u s e of
T4amB17, a n a m b er m u ta n t in g e n e 2 3 w h ic h l a c k s th e c o m p le te P23
p ro te in , w as a lso
u t i l i z e d to e s t a b l i s h th e p o s i t i o n o f t h i s b a n d in
th e p o ly a c r y la m id e g e l s .
C h lo ra m p h e n ic o l or RIF. tre a tm e n t o f c u ltu r e s d id n o t a l t e r the
14
19
c h a s i n g o f C - l e u c i n e from th e P23 b a n d by C - I e u c i n e (F ig . 18).
C u ltu r e s t r a n s i e n t l y t r e a t e d w ith b o th CAP a n d RIF a l s o 'a llo w e d C*4l e u c i n e to be c h a s e d from th e P23 b a n d (F ig s . 17 a n d 18). The r a d i o 12
a c t i v i t y in P23 w a s c o m p le te ly c h a s e d w ith C - l e u c i n e in u n tre a te d
c e l l s in. 10 m in u te s a n d e s s e n t i a l l y c o m p le te d in CAP a n d RIF t r e a te d
c e l l s in 20 m in u te s .
I n c o m p le te P2 3 p e p t i d e s from a m b e r m u ta n ts in g e n e 23 h av e
b e e n show n, to p o s s e s s c h a r a c t e r i s t i c m o b ilitie s in. p o ly a c r y la m id e
g e l s . T h is m o b ility is. in g e n e r a l i n v e r s e l y p ro p o r tio n a l to th e i r le n g th
(3 5 ,4 0 ). The u n c o m p le te d P23 p e p t i d e s from CAP t r e a t e d p o ly s o m e s
a re s h o w n .in F ig . 19. W h en th e CAP w a s rem o v ed from t h e s e c e l l s th e
in c o m p le te , p e p t i d e s w e re c h a s e d o u t o f p o ly a c r y la m id e g e l f r a c tio n s
12
4 - 8 in 10 m in u te s and. a p p e a r e d in f r a c tio n o n e . After a 2 0 m in u te C l e u c i n e c h a s e th e C ^ - I e u c i n e did n o t e n te r th e g e l s a t a l l .
14
A 15 s e c o n d p u l s e o f C - l e u c i n e in T4amN54 i n f e c t e d E . c o ll B
p ro d u c e d a d if f u s e r a d i o a c t i v e re g io n n e a r P2 3 b u t a f t e r a. 10 m inute
12
c h a s e w ith C - l e u c i n e a s h a rp w e ll d e fin e d b a n d a p p e a r e d a t the P23
p o s i t i o n . ( F i g . 2 0 ). C e l l s t r e a t e d w ith lO O p g /m l CAP $ t th e end o f th e
27
15 s e c o n d C 14- I e u c i n e p u l s e a l s o s h o w e d th e a b s e n c e o f th e P23 b a n d
IO
A fter re m o v a l o f th e CAP a n d a 2 O m inute c h a s e w ith C -^leucine th e
P2 3 b a n d a p p e a r e d .
The d a t a from t h e s e e x p e r im e n ts s u g g e s t t h a t th e n a s c e n t p e p ­
t i d e s w h o s e e lo n g a t io n h a s b e e n i n h i b i t e d b y CAP a re c o m p le te d and
r e l e a s e d n o rm a lly a f t e r re m o v a l o f th e a n t i b i o t i c .
% Inhibition of phage production
28
IO O
pg / ml
Fig u re I .
Chloramphenicol
C h lo ra m p h e n ic o l I n h ib itio n C u r v e . .E. c o li B w a s in f e c te d a t
3 0 ° C in M9 m e d ia w ith T4D w . t . a t a n M O I= 5. C h lo ra m p h e n ic o l w a s
a d d e d a t v a ry in g c o n c e n t r a t i o n s a t 2 min a n d gro w th c o n tin u e d to 90
min a f t e r in f e c tio n w h e n 0.5 ml s a m p l e s w e re c h ille d a n d t r e a t e d w ith
ch lo ro fo rm for 5 m i n . P h a g e t i t e r s w e re d e te rm in e d by th e a g a r la y e r
m e th o d (5 0 ).
29
-20
- 40
■
60
hibition of phggg production
-0
60
/ml
Figure 2.
Rifampicin
R ifam p icin I n h ib itio n C u rv e .
M eth o d w a s th e sam e a s
F ig . I , e x c e p t t h a t RIF w a s a d d e d a t v a ry in g c o n c e n t r a t i o n s a t 12 min
a f te r i n f e c t i o n .
30
Figure 3 .
C h lo ra m p h e n ic o l O ne Step G row th C u r v e .
E . c o li w as i n ­
f e c te d a t 3 0 ° C in M9 w ith an M O I= S a n d a f te r 5 m in s u p e r in f e c te d
w ith a n M O SI= 5. F ifty (ig/m l CAP w a s a d d e d a t 2 min a n d d ilu te d
1:100 w ith p re w arm ed M9 a t IOmin a f t e r i n f e c t i o n . S a m p le s of 0.5 ml
w e re re m o v e d a t th e i n d i c a t e d tim e s , c h i l l e d , t r e a t e d w ith chloroform
for 5 m in , a n d a s s a y e d b y th e a g a r la y e r m e th o d . C o n tro l s in g le b u r s t
c u rv e (O— O); 50 p g /m l CAP a d d e d a t 2 m in , d ilu te d 1 : 100 a t IO m in
( • — • ) ; a n d 50 p g /m l CAP a d d e d a t 2 min ( □ — □ ) .
50 ^jg/ml
CAP cidtibd
CAP dil.
50
Minutes After
Figure 3 .
Infection
C h lo ra m p h e n ic o l O n e Step G row th C u r v e .
32
50>jg/ml
RIF added
RIF dil.
I: IOO
V -------
Minutes
Fig u re 4 .
after infection
R ifam p icin O ne Step G row th C u r v e .
M e th o d w a s the sam e
a s F ig . 3 , e x c e p t t h a t 50 p g /m l RIF w a s a d d e d a t 12 m in a n d d ilu te d
w ith p re w arm ed M9 a t 18 min a f te r in f e c t i o n .
C o n tro l s i n g l e b u rs t
c u rv e (O— 0 ) ; 50 p g /m l RIF a d d e d a t 12 m i n , d ilu te d 1:100 a t 18 min
(S— • ) ; 50 p g /m l RIF a d d e d a t 12 min ( □ — D ) .
33
Fig u re 5 .
T4D w . t . P o ly s o m e s .
Ten ml o f E , p o ll B a t 3 0 ° C w ere i n ­
f e c te d w ith T4D w . t . a t a n MOI = 5 a n d s u p e r in f e c te d a t 5 min w ith an
M O SI= 5. At 15 min a f te r in f e c tio n 0.5 UCj C 14-Arg w a s a d d e d to a I min
p u l s e , c e l l s c h i l l e d , p e l l e t e d , a n d l y s e d by 2 c y c l e s o f f r e e z e - t h a w 2 min h e a ti n g w ith ly s o z y m e a s in " M a t e r i a l s a n d M e th o d s " . The
l y s a t e w a s s p u n for 2.2 5 h o u rs a t 25,000 rpm on 15-30% s u c r o s e g r a d i ­
e n t s , f r a c tio n s c o l l e c t e d , p r e c i p i t a t e d by co ld 5% TCA , and th e r a d i o ­
a c t i v i t y c o u n te d in a N u c le a r C h ic a g o p i a n e t t e c o u n te r . O ne min
C
- a r g p u ls e (O— O); 1 min C 14- a r g p u ls e fo llo w e d by 2 Ug/ml R N ase B
for 10 min a t 3 0 ° C ( # — • ) .
3000
Counts / minuts C Arginins
2000
1000
Fraction No.
Figure 5.
T4D w . t . P o l y s o m e s .
35
500
Fraction No.
Figure 6.
T4D w . t . Po ly so m e L y s is C o n t r o l s .
M eth o d w a s the sam e
a s F ig . 5 , e x c e p t for th e l y s i s p r o c e d u r e . The c e l l s w e re fro ze n in a
- 9 O0 C e th a n o l b a th a n d th a w e d in a O0 C i c e - w a t e r b a th for 2 c y c l e s .
Two c y c l e s f r e e z e - t h a w i n g o n ly ( 0 — O); f r e e z e - t h a w - h e a t i n g for 2 min
a t 3 0 ° C , 2 c y c l e s ( # — • ) ; f r e e z e - t h a w - h e a t i n g for 4 min a t SO0 C , 2
c y c le s (□ — □ ).
36
Fig u re 7.
T4D w . t . C h a s e d P o l y s o m e s . M eth o d w a s th e sam e a s F ig .
14
5 e x c e p t t h a t I pC i C - a r g w a s a d d e d for a I min p u l s e 2 Omin a f te r
i n f e c t i o n . O ne min C - a r g p u ls e (O— O); I min C ^ - a r g p u ls e fo llo w e d
by 2 p g /m l R N ase B for 10 min a t 3 0 ° C ( • —• ) ;
I min C 14- a r g p u ls e f o l -
12
lo w e d by IOmin c h a s e w ith 10 u g /m l C - a r g (□— Q ) .
2000
1500
o 1000
1 w _________ I_____________ i______________u
2
10
18
Fraction Mo.
Figure 7.
T4D w . t . C h a s e d P o ly s o m e s .
26
38
2000-
0»
1500-
C
C
P
<
O
« 1000 =I
C
c
3
O
O
500
10
18
Fraction No.
Fig u re 8
T4D w . t . C h a s e d P o l y s o m e s .
M eth o d w a s th e sam e a s F ig .
7. O ne min C ^ - a r g p u l s e fo llo w e d b y a IOmin c h a s e w ith 10 |ig /m l
C^2- a r g ( □ — □ ) ; o n e min C ^ - a r g p u l s e (O— O); I min C ^ - a r g p u ls e
fo llo w e d by 2 p g /m l R N ase B for 10 min a t 3 0 ° C ( ■ — ■ ) .
39
Fraction No.
Figure 9 .
R ifam p icin T re a te d T4D w . t . P o l y s o m e s . M e th o d w a s th e
14
s a m e a s F ig . 5 e x c e p t t h a t 2 pCi C - u r a c i l w a s a d d e d a t 20 min a f te r
i n f e c t i o n for I min in c o n tro l c e l l s a n d 5 min in RIF t r e a t e d c e l l s . O ne
min C^4-U ra c il p u l s e ( • — • ) ;
I min p u l s e fo llo w e d by a 2 tig /m l RN ase
B tr e a tm e n t for 10 min a t 3 O0 C (O— O); 100 |_ig/ml RIF a d d e d a t 17 min
fo llo w e d by a 5 min C 14-U ra c il p u l s e a t 20 min ( □ — □ ); 100 d g /m l RIF
a t 17 m i n , 5 min C 14-U ra c il p u ls e a t 20 min a n d a 2 p g /m l R N ase B
tr e a tm e n t a t 3 0 ° C for IOmin ( # — ■ ) .
40
IO O O
2
10
18
26
Fraction No.
Figure 10.
T r e a tm e n t.
P ro tein S y n th e s is After R ifam p icin and C h lo ra m p h e n ic o l
M eth o d w a s th e s a m e a s F ig . 5 e x c e p t 20 ml c u ltu r e s w ere
t r e a t e d w ith 100 d g /m l RIF a t 17 m i n , 10 d g /m l CAP w a s a d d e d a t 18 min
a n d th e c e l l s c h i l l e d a n d p e l l e t e d a t 2 0 min a f te r i n f e c t i o n . All
s a m p l e s w e re r e s u s p e n d e d in p re w arm ed M9 m edia a n d l a b e l e d w ith
I dCi C ^ - a r g for I min a t th e p ro p e r t i m e . RIF t r e a t e d c e l l s p u ls e d
w ith C 14- a r g a t 21 min (O— O); RIF a n d CAP t r e a te d c e l l s p u ls e d w ith
C*4- a r g a t 21 min a f t e r in f e c tio n (# —S).
41
2
IO
18
26
Fraction No.
Figure 11.
T re a tm e n t.
P ro te in S y n th e s is After R ifam p icin and C h lo ra m p h e n ic o l
M eth o d w a s th e sam e a s F ig . 10 e x c e p t t h a t th e I min p u ls e
w ith C 14- a r g o c c u r re d a t 30 min a f t e r i n f e c t i o n . RIF t r e a t e d c e l l s
p u l s e d w ith C i4- a r g a t 30 min (O— O); RIF a n d CAP t r e a t e d c e l l s p u ls e d
w ith C ^ - a r g a t 30 min a f te r in f e c tio n ( • — • ) .
42
IOOO
2
10
18
Fraction No.
26
Figure 12.
P ro te in S y n th e s is After R ifam p icin a n d C h lo ra m p h e n ic o l
T r e a tm e n t.
M e th o d w a s th e sam e a s F ig . 10 e x c e p t t h a t th e I min
p u l s e w ith C 14- a r g o c c u r r e d a t 40 m in a f t e r i n f e c t i o n . RIF tr e a te d c e l l s
p u l s e d w ith C 14- a r g a t 40 min (O— O); RIF and CAP t r e a t e d c e l l s p u l s e d
w ith C 14- a r g a t 40 min a f te r in f e c tio n ( # — # ) .
43
Fig u re 13 .
P ro tein S y n th e s is in T4amN54 P o ly s o m e s .
T w enty ml of
E . c o li B w e re in f e c t e d a t 3 0 °C w ith T4amN54 a t an M O I= 5 a n d s u p e r in f e c t e d a t 5 min w ith an M OSI= 5 . At 20 min a f te r in f e c tio n I pCi
14
C - l e u w a s a d d e d for 30 s e c o n d s , c e l l s c h i l l e d , p e l l e t e d a n d ly s e d
by 3 c y c l e s o f fr e e z in g a n d th a w in g w ith ly s o z y m e . The l y s a t e w a s
s p u n for 2.25 ho u rs a t 25,000 rpm on 15-30% s u c r o s e g r a d i e n t s , f r a c ­
tio n s c o l l e c t e d , p r e c i p i t a t e d by c o ld 5% TCA, a n d th e r a d i o a c t i v i t y
c o u n te d in a N u c le a r C h ic a g o p l a n c h e t t e c o u n te r . C o n tro l (O— O);
50 p.g/ml CAP a d d e d a t 20 min 30 s e c , w a s h e d ou t a t 2 2 min 30 s e c , and
12
c h a s e d for IOmin w ith 50 p g /m l C - l e u (D— □ ); sam e a s p re c e d in g
s a m p le , e x c e p t c h a s e d for 2 Omin w ith C
12
- l e u ( # —• ) .
Loucin
C
Counts / mi nut 3
Fraction
Figure 13.
No.
P ro te in S y n th e s is in T4amN54 P o l y s o m e s .
45
Fig u re 14.
P ro tein S y n th e s is in T4amN54 P o l y s o m e s . M eth o d w a s th e
s a m e a s F ig . 13. C
14
12
- l e u p u ls e c h a s e d w ith 50 iig /m l C - a r g for IOmin
a f t e r th e 30 s e c C - l e u p u ls e ( • — • ) ;
30 s e c C ^ - I e u p u l s e , t r e a te d
w ith 2 n g /m l R N ase B for IOmin a t 3 0 ° C ( □ — O ) .
5.000
Counts / minute
C
Leucine
10,000
IOOO
Fraction
Fig u re 14.
No.
P ro te in S y n th e s is in T4amN54 P o l y s o m e s .
47
Figure 15.
T4D P r o te in s in P o ly a c ry la m id e G e l s .
20 ml o f E. c o li B w ere
in f e c t e d a t 3 0 ° C w ith T4D or T4am Bl7 a t an MOI=S and s u p e r in f e c te d a t
an M O S I=S. At 20 min a f te r in f e c tio n I (iCi C ^ - I e u w a s a d d e d to a 2 min
p u lse.
The c e l l s w e re t h e n c h i l l e d , p e l l e t e d , a n d l y s e d b y 2 c y c le s o f
f r e e z e - t h a w - h e a t i n g w ith l y s o z y m e . U rea and B -m e rc a p to e th a n o l w ere
a d d e d to th e l y s a t e to m ake i t SM a n d 0.02M r e s p e c t i v e l y for th e s e i n ­
g r e d i e n t s . The u re a l y s a t e w a s s to r e d a t 4 ° C for 12 ho u rs , th e n la y e r e d
on a c i d i c SM u re a 7.5% p o ly a c r y la m id e g e ls , and the p r o te in s s e p a r a te d
by e l e c t r o p h o r e s i s .
T h e se g e l s w e re s t a i n e d w ith Amido S c h w a rz , d e -
s t a i n e d , s o a k e d for 6 h o u rs in 7% a c e t i c a c i d , s l i c e d lo n g itu d in a lly and
d r i e d . A u to ra d io g ra m s w e re m ade by e x p o s u r e o f the d rie d g e l s l i c e s to
X -ra y film for g e n e r a l l y 24 h o u r s . T he fo llo w in g l e t t e r s d e s i g n a t e s te p s
in v o lv e d in a s s a y i n g T4 p ro te in s w ith a c ry la m id e g e l s .
a_, g e l w ith Amido
S c h w a rz s t a i n e d T4Q p r o te in s ; b , d rie d lo n g itu d in a l s e c t i o n of g e l a;c , a u to ra d io g ra m o f r a d i o a c t i v e p r o te in s ; d , a n e l e c t r o p h o r e s i s d u p lic a te
o f g e l a; e_, dry lo n g itu d in a l s e c t i o n o f g e l d; t_, a u to ra d io g ra m of th e
r a d i o a c t i v e p r o te in s in g e l d.
48
Fig u re 15.
T4D P r o te in s in P o ly a c ry la m id e G e l s .
49
Figure 16.
A utoradiogram of T4D P2 3 H ead C o a t P ro te in . M eth o d w a s th e
14
sam e a s F ig . 15. C o n t . i s an u n in f e c te d C - l e u la b e l e d E . c o li B; B I7 is
T4amB17, a m u ta n t u s e d to l o c a t e P2 3 h e a d c o a t p r o te in . The follow ing
14
l e t t e r s d e s i g n a t e T4D i n f e c te d c e l l s p u ls e d w ith C - l e u for 2 min a t th e
i n d i c a t e d tim e s a f te r i n f e c t i o n , a , 10 min; b , 15 min; c , 20 min; d , 25 min;
£ , 3 0 min; f , 35 m in.
50
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Figure 17. A utoradiogram o f T4D P ro te in s R esu ltin g From Rifam picin and
C h lo ra m p h e n ic o l T r e a tm e n t. M eth o d w a s th e sam e a s F ig . 15 e x c e p t th a t
th e d e s i g n a t e d c e l l s w e re tr e a te d w ith 50 d g /m l RIF a t 17 m in , I pCi
C^4- I e u a t 20 m i n , a n d 50 p g /m l CAP a t 22 min a f te r i n f e c t i o n . At 24 min
th e c e l l s w e re c h i l l e d , p e l l e t e d , r e s u s p e n d e d , and c h a s e d w ith 50 d g /m l
C ^ -Ie u at 30°C .
B17 is T4amB17, a m u ta n t u s e d to i d e n tif y P23; a , 2 min
C ^ - p u l s e ; b , 2 min C ^ - p u l s e , 5 min C ^ - I e u c h a s e ; c , 2 min C ^ - I e u
p u l s e , IOmin C^2- I e u c h a s e ; d , 2 min C ^ - I e u p u l s e , 15 min C^2- I e u
c h a s e ; e , 2 min C ^ - I e u p u l s e , 20 min C^2- I e u c h a s e .
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Figure 18.
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50>ig/mlRIF@5Qug/ml CAP
A utoradiogram o f R ifam picin and C h lo ra m p h e n ic o l T reated T4D
P r o te i n s . M eth o d w a s the sam e a s F ig . 15. B I 7 i s T4amB17, a m utant
u s e d to id e n tif y P23; a , 2 min C 14- I e u p u ls e ; b , 2 min C 14- I e u p u ls e and
IOmin C - l e u c h a s e ; c , 2 min C 14- I e u p u ls e a n d 20 min C12- I e u c h a s e .
Cn
52
1200
800
-
9
10
Fraction No.
Fig u re 19.
P o ly a c ry la m id e G el F r a c tio n s Show ing C o m p le tio n of T4D
w . t . P e p tid e s A fter C h lo ra m p h e n ic o l R em o v al.
M e th o d w a s the sam e
a s F ig . 15, e x c e p t t h a t a t th e c o m p le tio n o f e l e c t r o p h o r e s i s th e p o ly ­
a c r y la m id e g e l s w e re im m e d ia te ly s l i c e d in to 0.5 cm s e c t i o n s , fin e ly
g r o u n d , a n d 1.0 ml o f h y d ro x id e o f h y a m in e a d d e d to e a c h s e c t i o n .
This w a s a llo w e d to s t a n d 12 h o u r s , th e n 10 ml o f s c i n t i l l a t i o n fluid
w a s a d d e d , a n d th e r a d i o a c t i v i t y c o u n te d in a N u c le a r C h ic a g o liq u id
s c i n t i l l a t i o n c o u n te r . A 2.5 |_iCi C 14- I e u p u ls e a t 20 min w ith 50 p g /m l
CAP a d d e d a t 22 min a f t e r in f e c tio n (£§&), C14- I e u p u l s e a t 20 min w ith
50 p g /m l CAP a d d e d a t 22 min a n d re m o v e d a t 24 min fo llo w e d by a
12
10 min 5 0 |a g /m l C - l e u c h a s e ( I
I). Same a s p r e c e d in g m ethod e x ­
c e p t c h a s e d for 20 min ( ■ ■ ■ ) .
53
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Figure 2 0 .
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A utoradiogram Show ing C o m p le tio n o f T4amN54 P e p tid e s After
C h lo ra m p h e n ic o l R e m o v a l. M eth o d w a s th e sam e a s F ig . 15 e x c e p t th a t
14
2.5 pC i C - l e u w a s a d d e d for a 15 s e c p u l s e in 20 ml in f e c te d c e l l s . a , 15
14
14
12
s e c C - l e u p u ls e ; b , 15 s e c C - l e u p u l s e , IOmin 50 d g /rn l C - l e u c h a s e ;
c , 15 s e c C14- I e u p u ls e fo llo w e d by 100 u g /m l CAP; d , s a m e a s p re c e d in g
m ethod b u t c e l l s c h i l l e d , p e l l e t e d , a n d r e s u s p e n d e d w ith o u t CAP and
12
c h a s e d w ith C - l e u for 20 m in.
DISCUSSION
T h e s e d a ta i n d i c a t e th a t CAP in h ib iti o n o f p o ly rib o s o m a l p ro te in
s y n t h e s i s i s c o m p le te ly r e v e r s i b l e a t th e mRNA r i b o s o m e - n a s c e n t
p e p tid e l e v e l .
M a tu re p h a g e fo rm atio n w a s found to re s u m e 7 m in u te s a f te r r e ­
m o v al o f CAP from T4D w . t . i n f e c t e d Ev c o li B a n d c u lm in a te d in a
n o rm a l b u r s t o f m a tu re in f e c tiv e p h a g e ( F i g . 3). This s h o u ld b e co m ­
p a re d to th e i r r e v e r s i b l e e f f e c t o f RIF w hich, p r e v e n ts a n y n e w p h a g e
fo rm atio n a f t e r i t s re m o v a l (F ig . 4 ). The d iag ram s h o w s a sm a ll
a m o u n t o f p h a g e fo rm a tio n w h e n RIF w a s a d d e d a t 12 m in u te s a f te r i n ­
f e c t i o n . This w o u ld be e x p e c te d if l a t e p h a g e f u n c t i o n s , s u c h a s P2 3
f o r m a ti o n , w e re a l r e a d y b e in g form ed a t 12 m in u te s . S upport for th is
w a s i n d i c a t e d by th e p re s e n c e , o f a s m a ll am o u n t o f P23 a t 10 m in u te s
a n d a n e a r m ax im a l am o u n t p r e s e n t a t 15 m in u te s a f t e r in fe c tio n , in
p o ly a c r y la m id e g e l s (F ig . 16). N e v e r t h e l e s s , '50 Ug/ml RIF in h ib ite d
fo rm a tio n of m a tu re T4D by 97% w h e n a d d e d 12 m in u te s a f t e r in f e c tio n
(F ig . 4).
The b a c t e r i o s t a t i c a c ti o n o f CAP on T4D s y n t h e s i s i s in a g r e e ­
m e n t w ith s t u d i e s c o n d u c te d on E . c o l i , S a lm o n e lla t y p h o s a , a n d o th e r
b a c t e r i a w h e re norm al g row th re s u m e d w h e n in h ib ito r y c o n c e n tr a tio n s
o f CAP w e re re m o v e d (58,'.59, 60). G row th of E . c o li a n d fo rm atio n o f
T4D e x h ib it th e s a m e s e n s i t i v i t y to CAP w h e re 10 p g /m l c o m p le te ly
55
i n h i b i t e d f u r th e r g ro w th . The c o m p le te r e c o v e r y a f t e r CAP tre a tm e n t of
T4D in f e c t e d E . c o li im p lie s, t h a t a n y c o n tin u e d .p r o te in s y n t h e s i s m u s t
in v o lv e p r e v i o u s l y in h ib ite d r i b o s o m e s . This fo llo w s from the know n
a b s e n c e o f fu r th e r h o s t.r ib o s o m e s y n t h e s i s a f t e r i n f e c t i o n a n d th e a b ­
s e n c e o f s i g n i f i c a n t l e v e l s of 70S m onom ers in th e T4D p o ly so m e
p re p a r a tio n s . (F igs . 5, 7 ,1 3 ).
The c o n tin u in g fu n c tio n o f CAP in h ib ite d ribosom e-m R N A com ­
p l e x e s a f t e r re m o v a l o f CAP w a s d e m o n s tr a te d by s u c r o s e d e n s it y
a n a l y s i s o f T4 w . t . a n d T4amN54 p o ly s o m e s .
N a s c e n t r ib o s o m a l p e p tid e s l a b e l e d w ith C"*^-l e u c i n e in
T4amN54 i n f e c t e d E , c o li w h ic h h a d b e e n t r e a t e d w ith 50 p g /m l CAP
12
w e re c h a s e d o u t of th e p o ly s o m e re g io n w ith C . - l e u c i n e ( F i g s . .13 z
and. 14).
This, r a d i o a c t i v i t y w a s 85% c h ase d , a f te r 10 m in u te s a n d 9-7%
12
c h a f e d , a f t e r 20 m in u te s w ith C - l e u c i n e . This p o ly s o m e r a d i o a c t i v i t y
a p p e a r e d in. th e s u p e r n a t a n t p o rtio n o f th e d e n s i t y g r a d ie n t a f te r th e
c h a se p e rio d s.
The s i m p l e s t e x p l a n a t i o n , a n d p e r h a p s th e m o s t e c o n o m ic a l for
th e c e l l , is t h a t n a t u r a l l y i n i t i a t e d mRNA-ribo.somal c o m p le x e s w ith
CAP tra p p e d in c o m p le te p e p tid e s a re a llo w e d to c o m p le te norm ally', be
r e l e a s e d , and. b e c o m e a s s o c i a t e d in to m atu re v iru s a f t e r re m o v a l of
th e a n t i b i o t i c . The d a t a d id n o t, h o w e v e r , ru le o u t th e p o s s i b i l i t y
t h a t t h e s e p e p t i d e s w e re n o t n o rm a lly c o m p le te d b u t m ig h t h a v e b e e n
56
r e l e a s e d in a n a b o r tiv e w a y . This r e l e a s e w o u ld be o n e s te p in f r e e ­
ing. b o u n d r ib o s o m e s s o th e y c o u ld r e i n i t i a t e on n e w mRNA.
The c o m p le tio n o f t h e s e i n h i b i t e d p e p tid e s w a s e x a m in e d by
u s in g o b s e r v a t i o n s o f H o s o d a a n d L e v in th a l (4 0 ). T hey found t h a t T4
g e n e 23 a m b e r m u ta n t p e p t i d e s m ig r a te d to d if f e r e n t p o s i t i o n s in p o l y ­
a c r y la m id e g e ls th a n d id th e w ild ty p e P23. In .g e n e r a l, t h i s m ig ra tio n
w a s i n v e r s e l y p ro p o r tio n a l to th e p e p tid e le n g th a n d w a s co n firm ed b y
L aem m li (3 5 ). As a r e s u l t , i t b e c a m e f e a s i b l e to lo o k a t p a r t i a l l y c o m ­
p l e t e d P23 p e p t i d e s on. p o ly a c r y la m id e g e l s . H o s o d a a n d L e v in th a l
(40) a l s o found t h a t c o m p le te d P2 3 s u b u n its a g g r e g a te e a s i l y in to
s t r u c t u r e s w h ic h m ig r a te d to th e to p o f th e s e p a r a t i o n g e l b u t d id n o t
e n t e r t h i s g e l . T h e s e a g g r e g a t e s w e re l a t e r in c o r p o r a te d in to m a tu re
phages.
14
In the. p r e s e n t s y s t e m CAP trap p ed . C - l e u c i n e l a b e l e d n a s c e n t
p e p t i d e s w e re s e p a r a te d , on a d e n s i t y g r a d i e n t , i s o l a t e d , and e x a m 12
i n e d in. p o ly a c r y la m id e g e ls (F ig . 19). After a 10 m in u te C - l e u c i n e
14
c h a s e th e u n c o m p le te d C - p e p t i d e s found in g e l f r a c tio n s 4 - 8 w e re
c o n v e r te d to a p r o d u c t l o c a t e d in g e l fr a c tio n o n e . W h e n a 20 m in u te
c h a s e w a s e m p lo y e d n e i t h e r th e p e p t i d e s in f r a c tio n s 4 - 8 nor th e
14
C - l a b e l in frac tio n , o n e w e re fo u n d in th e s e p a r a tio n , g e l s . T h e se r e ­
s u l t s a re c o n s i s t e n t w ith th o s e o f H o s o d a a n d L e v in th a l (40) and
L a e m m li.(35) a n d i n d i c a t e t h a t in c o m p le te P23 p e p t i d e s a re c o m p le te d
57
. a f t e r re m o v a l o f CAP a n d th e n a s s o c i a t e in to s t r u c t u r e s or a g g r e g a te s
w hich, a c c u m u la te a t th e s e p a r a t i o n g e l s u r f a c e .
The c o m p le tio n , o f CAP in h ib ite d P2 3 p e p tid e s w a s e x a m in e d b y a
s e c o n d m e t h o d . T4amN54 in f e c t e d E . c o li were, p u l s e d for 15 s e c o n d s
w ith C ^ - I e u c i n e in. o rd e r to produce, a large, p o p u la tio n o f u n c o m p le te d
12
l a b e l e d P23 p e p tid e s. (F ig . 2 0 ). C h a s in g w ith C - l e u c i n e p ro d u c e d a
r a d i o a c t i v e b a n d o f c o m p le te d P2 3 p r o t e i n s .
The c o n v e r s io n o f CAP
tr a p p e d P2 3 p e p t i d e s in to c o m p le te d P2 3 p ro te in a f t e r t r a n s i e n t t r e a t 12
■m e n t w ith CAP a n d a s u b s e q u e n t 2 0 m in u te C - l e u c i n e c h a s e d e m o n ­
s t r a t e d n o rm al c o m p letio n , for th e p e p t i d e s (F ig. 2 0 ).
I n h ib i te d T4D w . t . mRNA r e s u m e s i t s t r a n s l a t i o n a l fu n c tio n s
a f t e r th e re m o v a l of CAP. This c o n c l u s i o n w a s r e a c h e d b y in h ib itin g
RNA s y n t h e s i s w ith th e a n t i b i o t i c r if a m p i c in . This a llo w e d e x a m in a ­
tio n o f p r o t e i n - s y n t h e s i s d i r e c t e d b y mRN A m ade p rio r to th e a d d itio n
o f RIF a n d CAP ( F ig s . 10, 11, 12). The d a ta from th is e x p e r im e n t are
su m m arized, in T ab le I.
TABLE I:
R e c o v e ry o f P ro tein S y n th e s is in CAP In h ib ite d mRNA
( F i g s . 10,11, 12)
M in u te s F o llo w in g
CAP a n d RIF R em oval
P e rc e n t C o n tro l R ec o v e re d
RIF
RIF a n d CAP
I
100
. 11
10
64
63
20
0
54
58
The f i r s t c o l u m n . i n d i c a t e s th e tim e in m in u te s a f t e r rem o v al of
RIF a n d CAP from i n h i b i t e d c u ltu r e s . T h e s e c e l l s w e re p u l s e d for. I
m inutei w ith I p C i C " ^ -a rg in in e a t I , 10, a n d 2 0 m in u te s . C olum n two
i n d i c a t e s th e a b i l i t y o f RIF in h ib ite d , c u ltu r e s to c o n tin u e p ro te in s y n ­
t h e s i s a f t e r RNA s y n t h e s i s h a d b e e n s t o p p e d . The r a d i o a c t i v i t y i n c o r ­
p o r a te d a t o n e m in u te a f t e r th e re m o v e l o f RIF w a s s e l e c t e d a s the
14
100% c o n tr o l. The am o u n t of C - a r g i n i n e in c o r p o r a te d a t o th e r tim e s
a n d a f t e r CAP tr e a tm e n t (colum n 3) w e re r e l a t e d to t h i s c o n tro l a s a
p e rc en ta g e of i t .
The a b i l i t y o f RIF t r e a t e d c e l l s to, in c o r p o r a te C " ^ -a rg in in e
s t e a d i l y d e c r e a s e d u n til a t 2 0 m in u te s a f te r i t s re m o v a l no ra d io a c ^ '
t i v i t y w a s found in th e p o ly s o m e re g io n of th e d e n s i t y g r a d ie n t (F igs .
10, 11, 12 a n d T ab le I ) . The p ro te in s y n t h e s i s o c c u r rin g in CAP t r e a t e d
mRNA is m a rk e d ly d if f e r e n t th a n t h a t o c c u r rin g a f t e r RIF t r e a t m e n t .
14
The r a te o f C - a r g i n i n e in c o r p o r a tio n i n c r e a s e s d u rin g th e f i r s t 10
m in u te s a f t e r th e re m o v a l o f CAP b u t h a s d e c r e a s e d s l i g h t l y by 20 m in ­
u te s.
This i s i n t e r p r e t e d to m e an t h a t mRNA. p r e v i o u s l y in h ib ite d by
CAP r e s u m e s i t s a b i l i t y to c o d e n o rm a lly d u rin g p r o te in s y n t h e s i s a n d
in f a c t re m a in s f u n c tio n a l a f t e r mRNA n o t t r e a t e d w ith CAP h a s l o s t i t s
c a p a c i t y to in c o r p o r a te C ^ - a r g i n i n e . This w a s c o n s i s t e n t w ith r e ­
s u l t s o f o th e r w o rk e rs (62) w ho d e m o n s t r a t e d a p r o t e c t i v e a c tio n
im p a rte d to p o ly s o m e s by CAP.
59
An i n t e r e s t i n g o b s e r v a t i o n from t h e s e r e s u l t s i s t h a t C14a r g in in e p u l s e d in to T4D w . t . p o ly s o m e s w a s n o t c h a s e d , p u t w ith
12
. C - a r g i n i n e (F igs . 7 a n d 8). This r a d i o a c t i v i t y i s c o n v e r te d from a n
R N ase s e n s i t i v e s p e c i e s in to an R N ase r e s i s t a n t s p e c i e s a f te r th e - 10
12
m in u te C - a r g i n i n e c h a s e . T h e s e R N ase s e n s i t i v e a n d r e s i s t a n t
p e a k s w e re found to s e d im e n t to i d e n t i c a l regions, o f s u c r o s e d e n s i t y
g ra d ie n ts.
P a d a y a f ty a n d Rolfe (24) h a v e sh o w n t h a t P23 h e a d c o a t p ro te in
w a s. th e m a jo r c o m p o n e n t m ade on t h i s T4 p o ly s o m e re g io n la te in i n ­
f e c t i o n . Thus , th e .protein m a te r ia l found in th is R N ase r e s i s t a n t p e a k
i s l i k e l y to be h e a d c o a t p r o te in .(F ig s . 7 a n d 8).
An e x p la n a tio n .f o r th e d e v e lo p tn e lit o f th is p e a k m ay b e the o b ­
s e r v a t i o n o f Kiho a n d Rich (61) w ith 3 - g a l a p t o s i d a s e p o ly s o m e s o f
E. c o l i . They o b s e r v e d a n a g g r e g a tio n o f in d iv id u a l e n zy m e s u b u n its
in to a s tr u c tu r e o f 500,000 d a lto n s w h ic h d e m o n s tra te d e n zy m e a c t i v i t y
w h ile s t i l l a t t a c h e d to p o l y s o m e s . I t m ay be p o s s i b l e thpn. t h a t th e
i n i t i a l a g g r e g a tio n of P23 su b u n its; o c c u r s on th e h e a d c o a t p ro te in
p o ly s o m e . If th is i s t r u e , t h e s e a g g r e g a t e s p r o te c t th e mRNA of t h e s e
p o ly s o m e s from R N a s e . On. th e o th e r h a n d , th e p r e s e n c e of more c o m ­
p l e t e p h a g e c o m p o n e n ts s u c h a s e m p ty c a p s i d s h a s n o t b e e n e lim in ­
a t e d . This n o n c h a s a b l e R N ase r e s i s t a n t re g io n w a s e lim in a te d by
u s in g th e a m b er m u ta n t T4 a m N 5 4 . This p h a g e is m u ta n t in h e a d
a s s e m b l y g e n e 31 w hich, p r e v e n ts a g g re g atio n , o f P2 3. in to m ature p h a g e
60
h e a d s (6 3 ). I t w a s c o n s i s t e n t to fin d th a t a p h a g e m u ta n t in th is
a s s e m b l y g e n e did n o t d e v e lo p th e R N ase r e s i s t a n t p e a k found w ith
T4D w . t . i f th is r e s i s t a n t p e a k r e p r e s e n t e d a n a s s e m b l a g e of P23 s u b ­
u n its .
The w ork o f C am ero n a n d Ju lian . (18) on E . c o li Rel+ am ino a c id
a u x o tro p h s i n d i c a t e d th a t th e p o ly s o m e s o f th is s y s t e m r e g a in e d th e
a b i l i t y to in c o r p o r a te r a d i o a c t i v i t y w h e n CAP w a s re m o v e d . Their d e ­
c i s i o n w a s c a r e f u l ly q u a lif ie d b e c a u s e i t w a s im p o s s i b le to d i s t i n ­
g u i s h b e tw e e n n e w ly formed, la b e le d , n a s c e n t p e p tid e s and. n e w ly .. .
form ed l a b e l e d rib o s o m a l s tr u c tu r a l p r o te in s on CAP p a r t i c l e s co m ­
p le t e d a f te r re m o v a l o f CAP. It a p p e a r s from t h e s e T4 r e s u l t s th a t th e
re s u m e d a c t i v i t y in t h e i r p o ly s o m e s (18) p ro b a b ly a l s o in c lu d e d - th e
fo rm atio n o f n a s c e n t p e p tid e s..
G u r g o , A p ir io n , a n d S c h l e s s i n g e r (2 8) proposed, t h a t CAP and
S e v e ra l o th e r a n t i b i o t i c s u n c o u p le p o ly s o m e fo rm atio n from p e p tid e
b o n d f o r m a t i o n . This t h e y s a y a llo w s con tin u ed , rib o s o m e m ovem ent in
th e p r e s e n c e o f CA P. H o w e v e r , W e b e r a n d D eM o ss.(1 7 ) d e m o n s tra te d
t h a t CAP a llo w e d th e fo rm atio n of n e w p o ly s o m e s b u t n o t n a s c e n t p e p ­
t i d e s . M o rris a n d D e M o s s .(16) s h o w e d th a t p o ly s o m e s w e re formed
te n times, a s f a s t in am in o a c i d s t a r v e d Rel
a u x o tro p h s a fte r th e a d d i ­
tio n o f CAP th a n a f t e r a d d itio n o f th e re q u ire d am ino a c i d . F u r th e r ,
C am ero n a n d Julian. (18) d e m o n s t r a t e d t h a t 508 and, 308 rib o s o m a l s u b ­
u n its w e re c o n v e r te d to n o n f u n c tio n a l p o ly s o m e s w h ic h d id n o t c o n ta i n
61
. n a s c e n t p e p t i d e s in th e p r e s e n c e o f CAP. It w a s th e r e f o r e c o n c lu d e d
th a t CAP s to p p e d th e m o v e m e n t o f rib o s o m e s on mRNA. This w a s fu r­
t h e r s u p p o rte d b y th e d e m o n s tr a tio n t h a t am ino a c i d s t a r t e d Rel+ c e l l s
w h e n .tre a te d w ith CAP g e n e r a te d a norm al s e d im e n tin g , p o ly s o m e r e ­
gion, w ith o u t n a s c e n t p e p t i d e s te n tim e s a s f a s t a s w h e n th e p o ly ­
s o m e s w e re r e g e n e r a t e d n o rm a lly a f t e r th e re q u ire d am in o a c id w a s
added, to th e c u ltu re .
The p r e s e n t d a t a do n o t s u b s t a n t i a t e th e u n c o u p lin g m odel for
CAP p r o p o s e d b y G u r g o , A p irio n , a n d S c h l e s s i n g e r (2 8 ). S in c e u n ­
c o u p lin g w o u ld p r e s u m a b ly begin, im m e d ia te ly upon th e a d d itio n o f CAP
to a c u ltu r e o n e w o u ld e x p e c t th a t in c o m p le te d p ro te in frag m e n ts
w o u ld a c c u m u l a t e . If uncoupling, i s to a c c o u n t for th e r a p id r e g e n e r a tio n o f p o ly s o m e s in a n Rel
s t a r v e d c e l l a f te r a d d itio n of CAP i t m u s t
n e c e s s a r i l y be a f a s t p r o c e s s . If a n y a b n o rm a l r ib o s o m a l m ovem ent
o c c u r s w ith n o rm a lly i n i t i a t e d r ib o s o m e -mRNA c o m p le x e s upon the
a d d itio n o f CAP, th e n a s c e n t p e p t i d e s w o u ld n o t be e x p e c t e d to a p p e a r
a s norm al com pleted, p ro te in a f te r re m o v a l o f CAP. This i s c l e a r l y in
. o p p o s itio n to th e d e m o n s t r a t e d c o m p le tio n , of n o rm al P23 p ro te in a f t e r
CAP tr e a tm e n t in t h i s T4 s y s t e m .
SUMMARY
In h ib itio n o f p ro te in s y n t h e s i s b y c h lo ra m p h e n ic o l (CAP) w a s
c o m p le te ly r e v e r s i b l e on b a c te r io p h a g e T4 mRNA, n a s c e n t p e p tid e s ,
a n d h o s t E . c o li r ib o s o m e s a f t e r re m o v a l o f CAP. This w a s e s t a b l i s h e d
b y u s in g T4D w „ t. a n d T4 a m b er m u ta n ts amB17 a n d amN54 w ith v
d e n s i t y g r a d ie n t p o ly s o m e a n a l y s i s and. p o ly a c r y la m id e d i s c g e l
e lec tro p h o re sis.
The mRNA c o n tin u e s to c o d e for p ro te in s y n t h e s i s a f t e r t r a n s i e n t
tr e a tm e n t w ith CAP. T his fin d in g w a s d e m o n s tr a te d b y f i r s t t r e a tin g T4
i n f e c t e d c e l l s w ith rif a m p ic in to p r e v e n t fu rth e r mRNA s y n t h e s i s ,
w h ic h w a s fo llo w e d b y tr e a tm e n t w ith CAP to i n h i b i t p r o te in s y n t h e s i s .
The CAP w a s s u b s e q u e n t l y re m o v e d a n d th e u t i l i z a t i o n o f mRNA w h ic h
h a d b e e n form ed p rio r to r if a m p ic in tr e a tm e n t w a s t h e n t e s t e d by o b ­
s e r v in g th e r a t e o f C " ^ -a rg in in e i n c o r p o r a t i o n . This in c o rp o ra tio n i n ­
c r e a s e d s t e a d i l y d u rin g th e f i r s t 10 m in u te s a f te r CAP r e m o v a l , b u t 20
m in u te s l a t e r i t s r a te h ad d e c l i n e d o n ly s l i g h t l y . T his r e s u l t i n d i c a t e d
t h a t CAP t r e a t e d mRNA re m a in e d f u n c tio n a l a n d c o n tin u e d to c o d e for
p ro te in s y n t h e s i s a f t e r re m o v a l o f th e a n t i b i o t i c .
M e s s e n g e r R N A -ribosom e c o m p le x e s w h ic h a re s u b j e c t e d to CAP
tr e a tm e n t do re s u m e p ro te in s y n t h e s i s a f t e r re m o v a l o f th e a n t i b i o t i c .
N a s c e n t p e p t i d e s l a b e l e d w ith C ^ - I e u c i n e w ere tr a p p e d in th e p o ly ­
so m e re g io n o f a s u c r o s e d e n s i t y g r a d i e n t if CAP w a s a d d e d to T4-
63
i n f e c t e d c u ltu r e s . W ith t h e s e c e l l s C14- I e u c i n e c o u ld be c h a s e d , o u t
o f th e p o ly so m e, re g io n w h en C - l e u c i n e w as. a d d e d a f t e r CAP w a s - r e ­
m oved . E i g h ty - f iv e p e r c e n t o f th is l a b e l w a s removed, from th is re g io n
in a 10 m in u te c h a s e , in d i c a t i n g t h a t rib o s o m a l m o v e m e n t a n d fu n c tio n
. have been r e s to r e d .
N a s c e n t p e p t i d e s a re c o m p le te d a n d form n o rm al h e a d c o a t p r o ­
t e i n a f t e r t r a n s i e n t tr e a tm e n t w ith CAP. This w a s d e m o n s tr a te d on
p o ly a c r y la m id e g e l s b y th e a p p e a r a n c e o f th e h e a d c o a t p ro te in ban d
w h e n l a b e l e d n a s c e n t p e p tid e s in CAP t r e a t e d c e l l s w e re c o m p le te d
a f t e r th e re m o v a l o f CAP to form n o rm a l head, c o a t p r o t e i n .
This w a s d e m o n s tr a te d b y a s e c o n d m ethod: in c o m p le te h e a d
c o a t p e p t i d e s o f c e l l s t r a n s i e n t l y t r e a t e d w ith CAP w e re ch ase d , in to
th e p r o te in a g g r e g a te re g io n o f p o ly a c r y la m id e g e ls . T h erefo re ,
n a s c e n t p e p t i d e s in CAP t r e a t e d c u ltu r e s are c o m p le te d and. r e l e a s e d
n o rm a lly a f t e r re m o v a l o f th e a n t i b i o t i c .
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Mo n t a n a c -r.-^ r-_____
1
'VV10794 3
Mechanism of action
of chloramphenicol:...