Protein synthesis in an in vitro system of an extreme thermophile
by Clark Labach Gross
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE in Chemistry
Montana State University
© Copyright by Clark Labach Gross (1973)
Abstract:
The in vitro protein synthesizing system of T2, an unclassified gram-negative extremely thermophilic
bacteria, was investigated. The ribosomes of this organism are comparable to those of E. coli in
sedimentation constant. The incorporation of 14C-lysine was followed using a synthetic messenger,
polyadenylic acid. The optimal conditions for this incorporation of 14C-lysine have been determined.
This in vitro system has a temperature optimum for activity at 55°C. The level of incorporation of
14C-lysine with this system is similar to that of E. coli at 37°C. At higher temperatures, the Tg system
is much more efficient in synthesizing 14C-lysine peptides than the E. coli system. In p r e s e n t i n g t h i s t h e s i s in p a r t i a l fu lf illm e n t of th e re q u ire m e n ts
for a n a d v a n c e d d e g re e a t M o n ta n a S ta te U n i v e r s i t y , I a g r e e th a t th e
L ib rary s h a l l m ake i t f r e e ly a v a i l a b l e for i n s p e c t i o n .
I fu r th e r a g re e th a t
p e r m is s i o n for e x t e n s i v e c o p y in g o f th is t h e s i s for s c h o l a r l y p u rp o s e s
m ay be g r a n te d b y my m a jo r p r o f e s s o r , o r , in h is a b s e n c e , b y th e
D ir e c to r o f L i b r a r ie s .
It i s u n d e r s to o d t h a t a n y c o p y in g o r p u b lic a tio n
o f t h i s t h e s i s for f i n a n c i a l g a in s h a l l n o t be a llo w e d w ith o u t my w r itt e n
p e rm issio n .
S ig n a tu re
D ate
<^2 , / 9 " ? 3
PROTEIN SYNTHESIS IN AN IN VITRO SYSTEM OF
AN EXTREME THERMOPHILE
by
CLARK LAUBACH GROSS
■ 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 t i a l
f u lf illm e n t of t h e r e q u ir e m e n ts for th e d e g r e e
,
.
0,
MASTER OF SCIENCE
in
C h e m is tr y
A pproved:
df”, rJ if O
H e a d , M a jo r D e p a rtm e n t
<^-^Ct%ifman^ E xam W
Graduafte^D ean
MONTANA STATE UNIVERSITY
B o z e m a n , M o n ta n a
D ecem ber,
1973
iii
ACKNOWLEDGMENT
The a u th o r w i s h e s to th a n k :
D r. G o rdon R. J u lia n for c r e a t i n g th e o p p o rtu n ity for t h i s s t u d y ,
u n w a v e rin g s u p p o r t , a n d m an y h e lp fu l s u g g e s t i o n s ;
.
D r . J. T erry XJhlrich for i n v a l u a b l e h e lp in c u ltu r e a n d g ro w th of
th e r m o p h ili c b a c t e r i a ;
D r. John E. R obbins fo r a s s i s t a n c e in p h y s i c a l s t u d i e s ;
I
D r. S a m u e l J . R ogers fo r a d v ic e a n d h e lp fu l d i s c u s s i o n s ;
t h e C h e m is tr y D e p a rtm e n t for f i n a n c i a l s u p p o r t th ro u g h o u t th is
stu d y .
iv
TABLE OF CONTENTS
P ag e
ii
VITA ......................................................
ACKNOWLEDGMENT ..........................................................................
Hi
A B S T R A C T ................................................................
ix
1
INTRODUCTION
I
2
EXPERIMENTAL RATIONALE.................................................
8
3
MATERIALS AND M ETHODS....................................................................................10
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
( i)
B a c te r ia l s t r a i n s .........................................................................................10
B a c te r ia l g ro w th m e d i a ..................................................’
10
I n c u b a tio n c o n d i t i o n s ...........................................................
10
R ib o n u c le a s e r e m o v a l ........................................'................................ 11
B u f f e r s .............................................................................................................11
R e a g e n ts a n d c h e m i c a l s .............................
15
R ib o so m a l p r e p a r a t i o n ...............................................................................16
DEAE- c o lu m n p r e p a r a t i o n ............................................
18
P r e p a ra tio n o f pH 5 p r e c i p i t a b l e
p ro te in f r a c tio n . ............................................................................... 18
( j ) In v itr o in c o r p o r a tio n p r o c e d u r e s ...................................................... 19
(k) - C o u n tin g o f t o t a l a c i d - p r e c i p i t a b l e
c o u n t s ...................................................................................
20
( l ) S e d im e n ta tio n v e l o c i t y m e a s u r e m e n t s ........................................ 2 1
4
EXPERIMENTAL RESULTS....................................................................
(a)
(b)
(c)
(d)
(e)
R e q u ire m e n ts of PEP a n d p y r u v a te kinasein - ^ C - I y s i n e i n c o r p o r a t i o n ..............................
S e d im e n ta tio n p ro file o f T2 rib o s o m e s . . .
O p tim a l c o n d itio n s for in c o r p o r a tio n of
■■^C-lysine u s in g p o ly a d e n y l ic a c i d as
m e s s e n g e r a t ' 3 7 ° , 4 5 ° , 5 5 ° , a n d 6 5 °C . .
(1) M g++ t i t r a t i o n ........................................
(2) S p e rm id in e t i t r a t i o n .........................
(3) P o ly a d e n y lic a c i d t i t r a t i o n . . .
I n c o rp o ra tio n of 1 4 C - I y s i n e c o m p a re d a t
3 7 ° , 4 5 ° , 5 5 ° , a n d 6 5 °C . . .........................
Time s tu d y of in c o r p o r a tio n o f 14C - I y s i n e a t
37° 4 5 ° , 5 5 ° , and 65°C ..................................
22
22
22
.
.
.
.
22
23
23
23
.
23
24
V
Pag e
( f ) Time s tu d y o f in c o r p o r a tio n o f ^ C - I y s i n e a t
3 7 ° , 4 5 ° , 5 5 ° , a n d 6 5 °C u s in g th e E. c o li
s y s t e m .................... ............................. .... . .............................................24
5
D I S C U S S I O N ........................................................................................................„ 2 5
6
TABLES.............................................................................................. .
7
FIGURES ............................................. ' ........................................................................36
8
LITERATURE CITED
33
82
vi
LIST OF TABLES
Page
TABLE
14
1
O ptim um c o n d itio n s for in c o r p o r a tio n o f
2
Tg r ib o s o m a l s e d i m e n t a t i o n c o n s t a n t s
3
I ^ Q - I y s i n e in c o r p o r a tio n in p r e s e n c e a n d a b s e n c e
o f ATP g e n e r a t i n g s y s t e m .....................................................................
.
.
C -Iy sin e
.
. . .. . .3 3
.............................................34
.3 5
v ii
LIST OF FIGURES
P ag e
FIGURE
1
S e d im e n ta tio n p ro f ile o f T^ r i b o s o m e s ................................................. 37
2
M g++ t i t r a t i o n a t 3 7 ° C ± 4 mM s p e r m i d i n e .............................
3
M g++ t i t r a t i o n a t 4 5 ° C ± 4 mM s p e r m id in e .............................................. 41
4
M g++ t i t r a t i o n a t SS0 C ± 4 mM s p e r m i d i n e .............................................. 43
5
Mg++ t i t r a t i o n a t 6 5 °C ± 4 mM s p e r m id in e .............................................. 45
6
S p e rm id in e t i t r a t i o n a t 3 7 ° C ........................................................................... 47
7
S p e rm id in e t i t r a t i o n a t 4 5 ° C
8
S p e rm id in e t i t r a t i o n a t SS0 C ...........................................................................SI
9
S p e rm id in e t i t r a t i o n a t 6 5 °C .
. .
39
...........................................................................49
.
.................................................................S3
10
P o ly a d e n y lic a c i d t i t r a t i o n a t 3 7 ° C
.
...........................
55
11
P o ly a d e n y lic a c i d t i t r a t i o n a t 4 5 ° C ......................................................
12
P o ly a d e n y lic a c i d t i t r a t i o n a t SS0 C .............................................................59
13
P o ly a d e n y lic a c i d t i t r a t i o n a t 6 5 ° C .............................................................SI
14
In c o rp o ra tio n of ^ C - I y s i n e a t 3 7 ° , 4 5 ° , 5 5 ° a n d SS0 C
. . .
63
15
Time s tu d y o f in c o r p o r a tio n o f ^ C - I y s i n e a t 3 7 ° C
w ith Tg s y s t e m ................................................................................................
65
57
16
Time s tu d y o f in c o r p o r a tio n o f ^ C - I y s i n e a t 4 5 ° C
w ith Tg s y s t e m .......................................................................................................67
17
Time s tu d y o f in c o r p o r a tio n of ^ C - I y s i n e a t SS0 C
w ith Tg s y s t e m .................................................................................................
18
Time s tu d y o f in c o r p o r a tio n of ^ C - I y s i n e a t 6 5 °C
w ith T s y s t e m , .....................................
2
69
71
v iii
FIGURE
Page
19
Time s t u d y o f in c o r p o r a tio n o f ^ C - I y s i n e a t 3 7 ° ,
4 5 ° , 5 5 ° , a n d 6 5 ° C w ith E. c o l i ........................................................... 73
20
C o m p a ris o n o f T2 a n d E. c o li s y s t e m s a t 3 7 ° C . . . . . . . .
21
C o m p a ris o n of Tg a n d E_. c o li s y s t e m s a t 4 5 ° C .................................... 77
22
C o m p a ris o n of Tg a n d E. c o li s y s t e m s a t 5 5 °C . . . . . . .
23
C o m p a ris o n o f Tg
75
79
a n d Ev c o li s y s t e m s a t 6 5 ° C .................................... 81
ix
ABSTRACT
The in v itr o p ro te in s y n t h e s i z i n g s y s t e m of TV,, a n u n c l a s s i f i e d
g r a m - n e g a t i v e e x tr e m e l y th e rm o p h ilic b a c t e r i a , w a s in v e s t ig a te d .. The
r ib o s o m e s o f t h i s o rg a n is m a re c o m p a r a b le to th o s e of Tb c o li in s e d i ­
m e n ta tio n c o n s t a n t . The in c o r p o r a tio n o f -*-4C - l y s i n e w a s fo llo w e d u s i n g
a s y n t h e t i c m e s s e n g e r , p o l y a d e n y l i c a c i d . The o p tim a l c o n d itio n s for
t h i s in c o r p o r a tio n o f l ^ C - l y s i n e h a v e b e e n d e te r m in e d .
This in v itr o s y s te m h a s a te m p e r a tu r e optimum for a c t i v i t y a t
5 5 ° C . The l e v e l of in c o r p o r a tio n of - ^ C - I y s i n e w ith t h i s s y s te m is
s i m i l a r to t h a t o f .E. c o li a t 3 7 ° C . At h ig h e r t e m p e r a t u r e s , th e Tg s y s te m
i s m uch more e f f i c i e n t in s y n t h e s i z i n g 1^ C - I y s i n e p e p t i d e s th a n th e E .
c o li s y s t e m .
I
INTRODUCTION
R e p ro d u c tio n a n d g ro w th o f liv in g o rg a n is m s a r e in f l u e n c e d b y a
v a r i e t y of e n v ir o n m e n ta l f a c t o r s . ■ T e m p e ra tu re i s one o f t h e e a s i e s t
v a r i a b l e s to m e a s u r e .
The h ig h te m p e r a tu r e e n v iro n m e n ts e x e m p lifie d by
h o t s p r in g s in Y e llo w s to n e N a tio n a l P a rk , I c e l a n d , N ew Z e a l a n d , J a p a n ,
a n d R u s s ia p ro v id e u n iq u e o p p o r tu n i tie s to s tu d y th e e f f e c t o f te m p e ra tu re
on th e d i s t r i b u t i o n a n d m e ta b o lis m of liv in g o r g a n i s m s .
P r o c a r y o tic o r g a n i s m s , s u c h a s b l u e - g r e e n a l g a e a n d b a c t e r i a ,
i n h a b i t th e s e , th e rm a l s p r i n g s .
The te m p e r a tu r e o f t h e e n v iro n m e n t, a s
w e ll a s p H , s a l i n i t y , n u t r i e n t s , a n d li g h t i n t e n s i t y d e te r m in e w h ic h
o rg a n is m w i l l p o p u la te a g iv e n s p r i n g .
Brock (I) h a s s h o w n t h a t th e
te m p e r a t u r e optim um for b l u e - g r e e n a l g a e h a s a d e f in ite u p p e r lim it of
75 C .
H o w e v e r, b a c t e r i a c a n b e fo u n d g ro w in g in p o o ls in Y e llo w sto n e
N a tio n a l. Park w h e re th e w a t e r is a c t u a l l y s u p e r h e a t e d .
There a re m an y t h e o r i e s to e x p la i n th e e x i s t e n c e o f o rg a n is m s
liv in g in s u c h e x tre m e e n v iro n m e n ts a n d t h e y c a n b e d iv id e d in to tw o
g e n e r a l c a t e g o r i e s (2 ).
The f i r s t c a te g o r y c o n c e r n s th e r e p u te d a b i l i t y of th e o rg a n is m to
r a p id ly r e s y n t h e s i z e a n y d a m a g e d c o m p o n e n t of th e c e l l a n d t h e r e f o r e ,
re m a in e s s e n t i a l l y u n d a m a g e d .
T his g e n e r a l th e o ry d o e s n o t s e e m to
h o ld s i n c e a n o rg a n is m g ro w in g a t 7 0 °C s h o u ld t h e o r e t i c a l l y be a b le to
s y n t h e s i z e c e l l u l a r c o m p o n e n ts a t a r a t e of 16 tim e s t h a t a t 3 0°C
a s s u m in g a d o u b lin g o f r e a c t i o n r a te for e v e r y 10° r i s e in te m p e r a t u r e .
This a c c e l e r a t i o n o f lif e p r o c e s s e s h a s n e v e r b e e n s h o w n .
2
The s e c o n d g e n e r a l c a t e g o r y in f e r s t h a t th e c e l l u l a r c o m p o n e n ts o f
a th e r m o p h ili c o rg a n is m a re in h e r e n t l y more h e a t s t a b l e t h a n th e co m ­
p o n e n ts' o f m e s o p h i l i c o r g a n i s m s .
M o s t o f th e w ork c o n c e r n in g th is
c a t e g o r y d e a l t w ith i s o l a t i o n a n d s tu d y of c e l l u l a r c o m p o n e n t s .
G a u g h ra n (3) p o s t u l a t e d t h a t th e c o m p o s itio n o f l i p i d s in a th e rm o ­
p h i l i c b a c t e r i a m ay d i c t a t e th e te m p e r a tu r e lim it of g ro w th s i n c e a n im a ls
liv e a t t e m p e r a t u r e s b e lo w t h e i r lip id m e ltin g p o in ts .
T his th e o ry h a s
b e e n i n v e s t i g a t e d by a n u m b e r o f a u th o r s w ith c o n f lic tin g r e s u l t s .
Some
o f t h i s w o rk w i l l b e b r ie f ly m e n tio n e d b e lo w .
B aum an a n d Sim m onds (4) i n v e s t i g a t e d th e d i s t r i b u t i o n . o f lip id s in
a F l e x i b a c t e r i a , a n e x tre m e th e rm o p h ile w h ic h g ro w s in f ila m e n to u s
b acte ria l m a s s e s .
C ^g c o m p a r e d to
A lth o u g h th e g r e a t e s t p e r c e n t a g e o f f a t t y a c i d s w e re
in m e s o p h i l i c b a c t e r i a , t h e y c o n c lu d e d t h a t th e
d i s t r i b u t i o n w a s n o t r e m a r k a b le .
D aro n (5) i n v e s t i g a t e d th e d i s t r i b u t i o n o f lip id s in a th e rm o p h ilic
s tra in of B a c illu s .
He c o n c lu d e d t h a t th e p ro p o rtio n o f u n s a t u r a t e d f a tty
a c i d s v a r i e d i n v e r s e l y w ith th e tem . o ra tu re a n d th e g r e a t e s t p e r c e n ta g e
o f t h e s e f a t t y a c i d s w a s 14%.
H o w e v e r, th e p ro p o rtio n o f u n s a t u r a t e d
f a tty a c i d s d e p e n d e d g r e a t l y on th e e n e r g y s o u r c e .
U n f o r t u n a t e l y , th e r e h a s n o t b e e n e n o u g h e x p e r im e n t a tio n in lip id
r e s e a r c h to d e te r m in e i f th e m em brane i s r e s p o n s i b l e fo r th e th e rm a l
r e s i s t a n c e o f t h e s e o r g a n is m s .
A n o th er p l a u s i b l e e x p l a n a t i o n for th e rm o p h ily is t h a t th e DNA of
3
t h e s e o r g a n is m s a re more r e s i s t a n t to h e a t .
S in c e th e m e ltin g p o in t of
DNA i s r e l a t e d to th e G -C c o n t e n t , i t i s c o n c e i v a b l e t h a t th e rm o p h ile s
c o u ld h a v e a h ig h e r G - C c o n t e n t t h a n m e s o p h i l e s .
Brock a n d F re e z e (6)
h a v e sh o w n t h a t a n e x tre m e t h e r m o p h i l e , T. a g u a t i c u s , h a s a G -C c o n ­
t e n t of 65%.
On th e o th e r h a n d , B . s te a r o th e r m o p h ilu s h a s a G -C
c o n t e n t o f 44% .
26.5% to 74% ( 7 ) .
M e s o p h i l i c b a c t e r i a h a v e G -C c o n te n t s ra n g in g from
T h e r e f o r e , th e G - C c o n te n t i s u n lik e ly to be r e s p o n ­
s i b l e for th e r m a l s t a b i l i t y .
A c u r r e n t fie ld o f i n t e n s i v e s tu d y h a s b e e n th e r m o p h ilic p ro te in s
a n d t h e i r p r o te in s y n t h e s i z i n g m a c h i n e r y .
M o s t of th e e x p e r im e n t a tio n
h a s b e e n lim ite d to th e s p o r e - f o r m in g th e rm o p h ilic s p e c i e s of B a c i l l u s .
M u ch o f th e e a r l y w ork w a s r e s t r i c t e d to s t r u c t u r a l p r o te in s s u c h
a s th e f l a g e l l a of B a c illu s (2 ).
T h e s e f l a g e l l a e s h o w e d m uch more h e a t
s t a b i l i t y t h a n f l a g e l l a e from m e s o p h i lic s t r a i n s a n d w e re more r e s i s t a n t
to c l a s s i c a l d e n a tu r in g a g e n t s s u c h a s u r e a , a c e t a m i d e , a n d sodium
dodecyl s u lfa te .
L a te r e x p e r im e n ts c o m p a re d th e p h y s i c a l p r o p e r tie s of p u rifie d
e n z y m e s from th e r m o p h ili c a n d m e s o p h i lic b a c t e r i a .
M a n n in g and
C a m p b e ll (8) c r y s t a l l i z e d a - a m y l a s e from B_. s te a r o th e r m o p h i lu s a n d
d e te r m in e d th e optimum te m p e ra tu re for a c t i v i t y to b e in th e 5 5 °C to
7 0 °C r a n g e .
The m o le c u la r w e ig h t w a s d e te r m in e d a s 1 5 ,6 0 0 d a lto n s
(9) c o m p a re d to 4 8 , 6 5 0 d a lto n s for a - a m y l a s e i s o l a t e d from a m e s o p h ilic
s p e c i e s , B_. s u b t i l u s . A n o th e r s tu d y (10) s h o w e d th a t t h e th e rm o p h ilic
4
a - a m y l a s e c o n ta i n e d a 4. 5 fo ld e x c e s s o f pro lin e c o m p a re d to th e m e s o Phi l i e e n z y m e ^ The d is o r d e r e d s tr u c tu r e r e s u l t i n g from th e h ig h p ro lin e
c o n t e n t w a s p o s t u l a t e d a s t h e r e a s o n for th e th e rm a l s t a b i l i t y .
M a n y o th e r i s o l a t e d e n z y m e s h a v e b e e n s t u d ie d a n d a lth o u g h th e r e
m ay b e som e e x c e p t i o n s , m o s t of th e e n z y m e s from therm op h ile s are
m uch more r e s i s t a n t to h e a t th a n th e c o r r e s p o n d in g m e s o p h i lic e n z y m e s .
S in c e a - a m y l a s e i s s u c h a s m a ll p r o t e i n , i t h a d b e e n th o u g h t t h a t
la r g e r e n z y m e s w o u ld n o t sh o w h e a t r e s i s t a n c e .
S tu d ie s o f g l y c e r a l d e -
hy d e 3 - p h o s p h a t e d e h y d r o g e n a s e (11) ( M . W . = 1 3 0 ,0 0 0 d a lto n s ) from
B. s t e a r o th e r m o p h i lu s s t i l l s h o w r e s i s t a n c e to h e a t . ' The c o m p a r is o n o f
t h i s e n z y m e to m e s o p h i lic e n z y m e s in a m in o a c id c o m p o s itio n d o e s n o t
sh o w n a n y s i g n i f i c a n t d if f e r e n c e s a lth o u g h th e p rim ary s e q u e n c e is n o t
know n.
The l i t e r a t u r e in t h i s f ie ld is b e c o m in g r e p l e t e w ith e x a m p le s of
t h e r m o s ta b le e n z y m e s . A lth o u g h th e r e a s o n for th is s t a b i l i t y i s unknow n,
the. a p p l i c a t i o n o f s e q u e n c e a n a l y s i s a n d X - r a y c r y s t a l l o g r a p h y w ill
p ro v e v a l u a b l e in d e te r m in in g th e p rim ary s tr u c tu r e to i n d i c a t e th e ty p e s
o f i n t e r a c t i o n s n e c e s s a r y to c o n fe r th e rm a l s t a b i l i t y . ,
W ith th e i n t e r e s t g e n e r a t e d in th e rm o p h ilic p r o t e i n s , i t w a s o n ly
n a tu r a l t h a t th e p ro te in s y n t h e s i z i n g m a c h in e ry s h o u ld b e i n v e s t i g a t e d
on a s u b c e l l u l a r l e v e l .
The f i r s t re p o r ts (12) of t h e r m o s ta b le am ino
a c i d - a c t i v a t i n g e n z y m e s s u g g e s t e d t h a t th e rm o p h ilic s y s t e m s c o u ld be
u t i l i z e d in th e s tu d y o f c o d in g p r o p e r tie s of m e s s e n g e r RNA a n d in
5
t e m p e r a t u r e - i n d u c e d m o d if ic a tio n s of t h i s m e s s e n g e r .
F ried m an a n d W e i n s t e i n (13) u s e d a s u b c e l l u l a r s y s te m from B_.
stea ro tb e rm o p h ilu s.
14
They w e re a b le to d e m o n s tr a te t h a t in c o r p o r a tio n o f
o
C ^-labelled p h e n y l a l a n i n e , l y s i n e , a n d p ro lin e w a s o p tim a l a t 55 C
to 6 0 ° C u s i n g a n a tiv e m e s s e n g e r RNA.
They a l s o s h o w e d t h a t in c o r p o r ­
a t i o n o f - ^ C - I y s i n e w a s g r e a t e r a t 6 5 °C th a n a t 3 7 °C u s in g s y n t h e t i c
p o ly ad en y lic a c id as m e sse n g e r.
This g r e a t l y i n c r e a s e d in c o rp o ra tio n
w a s p o s t u l a t e d a s th e e n h a n c e d a b i l i t y of a tta c h m e n t o f m e s s e n g e r to
th e r ib o s o m e s b e c a u s e o f th e l o s s o f s e c o n d a r y s tr u c tu r e of th e m e s s e n ­
g e r a t th e h ig h e r t e m p e r a t u r e .
By c o m p a r is o n , a n E . c o li s y s te m sh o w e d
o n ly 10% i n c o r p o r a tio n a t 6 5 ° r e l a t i v e to b a s e lin e l e v e l s a t 3 7 ° C .
T h e s e a u th o r s a l s o d e m o n s t r a t e d t h a t th e th e rm a l d e n a t u r e tio n p ro file
o f t-RNA from B_. s te a r o tb e r m o p h i lu s w a s n o t s i g n i f i c a n t l y d if f e r e n t
from t h a t o f E . c o l i .
A lg ra n a ti a n d L e n g y e l (14) a l s o i n v e s t i g a t e d a s u b c e l l u l a r s y s te m
from B_. s te a r o tb e r m o p h i lu s b u t e m p lo y e d a random c o p o ly m e r of a d e n y li c
a n d u r id y l ic a c i d .
T h ey w e re i n t e r e s t e d in d e te r m in in g how m uch i n c o r ­
p o r a tio n w o u ld o c c u r a t h ig h e r te m p e r a t u r e b e c a u s e of th e h ig h d e g re e
o f s e c o n d a r y s t r u c t u r e in t h i s c o p o ly m e r.
They found t h a t t h i s s y n t h e t i c
m e s s e n g e r w a s c o m p le te ly i n a c t i v e in th e E_. c o li s y s t e m b u t had som e
a c t i v i t y a t 3 7 ° C in th e th e r m o p h ilic s y s t e m .
This a c t i v i t y w a s i n c r e a s e d
3 - f o l d w h e n in c u b a t e d a t 6 5 ° C .
In a d d i t i o n to h e a t r e s i s t a n t a m in o a c i d - a c t i v a t i n g e n z y m e s .
6
th e r m o p h ilic b a c t e r i a a l s o p o s s e s s h e a t r e s i s t a n t r i b o s o m e s .
Friedm an
(15) p r e h e a t e d E_.. c o li a n d B,. s te a r o th e r m o p h i lu s r ib o s o m e s for 15 m in ­
u tes a t 65 °C .
He th e n t e s t e d th e a b i l i t y o f t h e s e r ib o s o m e s to in c o r p o r ­
a t e ^ ^ C - p h e n y l a l a n i n e a t 3 7 ° C u s in g p o ly u r i d y lic a c id a s m e s s e n g e r .
W ith t h i s p re t r e a t m e n t , E . c o li r ib o s o m e s c o u ld in c o r p o r a te o n ly 5% of
t h a t o b s e r v e d a t 3 7 ° C w ith no p r e tr e a tm e n t .
The r ib o s o m e s o f B .
s te a r o th e r m o p h i lu s w e re v i r t u a l l y u n a f f e c t e d s i n c e th e y in c o r p o r a te d
80% o f t h a t o b s e r v e d a t 3 7 ° C w ith no p re t r e a t m e n t .
E x t e n s iv e s tu d y of th e rm a l d e n a tu r a ti o n p ro f ile s of rib o s o m e s b y
P a c e a n d C am p b ell(IG ) d e m o n s t r a t e d t h a t th e rib o s o m e s of th e rm o p h ile s
h a d a m uch h ig h e r m e ltin g p o in t th a n t h o s e o f m e s o p h i l e s .
This o b s e r ­
v a t i o n le d th e a u th o r s to s p e c u l a t e t h a t th e m e ltin g p o in t o f th e rib o so m e
m ay s e t th e u p p e r lim it of g r o w t h .
The v a s t m a jo rity o f th e r e s e a r c h on th e rm o p h ilic o r g a n is m s h as
u t i l i z e d s p o r e - f o r m in g th e r m o p h ilic s p e c i e s o f B a c i l l u s .
R e c e n tly , a
n o n - s p o r u l a t i n g e x tre m e th e rm o p h ile w h ic h i s g r a m - n e g a tiv e h a s b e e n
d e s c r i b e d b y Brock a n d F r e e z e (6 ).
This o rg a n is m w a s o r i g i n a l l y i s o ­
l a t e d from Y e llo w s to n e N a tio n a l Park a lth o u g h i t c a n b e fo u n d in s u c h
d i v e r s e s p o t s a s th e ho t w a t e r h e a t e r s o f c o m m e rc ia l la u n d ro m a ts (1 7 ).
This o rg a n is m h a s b e e n c l a s s i f i e d Therm us a q u a t i o ns a n d i t i s an
o b lig a te a e r o b e w ith a n optimum g ro w th te m p e ra tu re of 7 0 o C.,
Z e ik u s e t a l . (1.8) h a v e i n v e s t i g a t e d som e of th e p h y s i c a l and
c h e m i c a l p r o p e r tie s o f th e rib o s o m e s a n d RNA from t h i s o rg a n is m .
The
7
r ib o s o m e s c o n t a i n 59% p r o te in a n d 41% RNA c o m p ared to 59% RNA a n d
41% p r o te in in B . s te a r o th e r m o p h i lu s b u t t h i s fin d in g m ay r e f l e c t d if f e r ­
en t cu ltu ra l c o n d itio n s.
The th e rm a l d e n a tu r a ti o n p ro file o f rib o s o m e s
e x h i b i t e d a m e ltin g te m p e r a tu r e o f SG0 C a n d th e a u th o r s c o r r e la te th e
te m p e r a tu r e lim it o f g ro w th w i t h t h i s m e ltin g p o in t.
F u rth e r i n v e s t i g a t i o n b y S te llw a g e n (19) h a s sh o w n t h a t th is o r g a n ­
is m p o s s e s s e s e x tr e m e ly t h e r m o s ta b le e n z y m e s .
He s t u d i e d e n o l a s e
from T. a g u a t i c u s a n d found th a t th e e n z y m e w a s c o m p o s e d o f 8 e q u a l
p o ly p e p tid e c h a in s a n d h ad o p tim a l a c t i v i t y a t 9 0 ° C .
A bove 9 0 ° , th e
enzym e rap id ly d e n a tu r e d .
F r e e z e (20) h a s a l s o sh o w n t h a t f r u c to s e d ip h o s p h a t e a l d o l a s e from
T. a g u a t i c u s h a s a te m p e r a tu r e optim um o f 9 0°C b u t he w o rk e d w ith a
p a r t i a l l y p u rif ie d e n z y m e .
A lth o u g h e n z y m e s a n d rib o s o m e s from T. a g u a t i c u s h a v e b e e n i s o ­
l a t e d , th e r e h a v e b e e n no re p o rts o f p ro te in s y n t h e s i s in a s u b c e l l u l a r
sy ste m .
This s y s te m w ill u n d o u b te d ly b e c la r if ie d in a fe w y e a r s .
2
EXPERIMENTAL RATIONALE
M o s t i n v e s t i g a t i o n s on th e p r o te in s y n t h e s i z i n g m a c h in e ry of
th e r m o p h ili c b a c t e r i a h a v e b e e n lim ite d to s p o r e - f o r m i n g , g r a m - p o s i t i v e
o r g a n is m s s u c h a s B. s t e a r o t h e r m o p h i l u s .
The r e s u l t s of t h e s e i n v e s t i ­
g a t i o n s h a v e b e e n c o m p a re d to E . c o l l , a g r a m - n e g a tiv e b a c t e r i a common
to th e lo w e r d i g e s t i v e t r a c t of m a n .
R e c e n tly , a n e x tr e m e l y th e r m o p h ilic b a c t e r i a d e s i g n a t e d a s Tg w a s
i s o l a t e d b y U h lr ic h (21),.
This o r g a n i s m , a lth o u g h u n c l a s s i f i e d , r e s e m ­
b l e s T. a g u a t i c u s w h ic h w a s i s o l a t e d b y Brock (6 ).
It i s g r a m - n e g a tiv e
a n d n o n - s p o r u la tin g a n d form s lo n g f il a m e n ts a t h ig h t e m p e r a t u r e s .
This o rg a n is m w a s found to be in d u c ib le for 3 - g a l a c t o s i d a s e (2 1 ),
a n in d u c ib le e n z y m e w h ic h h a s b e e n - w e ll c h a r a c t e r i z e d .
This en zy m e
w a s in d u c e d b y l a c t o s e a n d r e p r e s s e d b y g l u c o s e in th e c l a s s i c m a n n er
(2 2 ).
H o w e v e r , t h i s e n z y m e w a s th e r m o s t a b l e a n d e x h i b i t e d m ax im al
a ctiv ity at 7 0 ° C .
S in c e t h i s e x tre m e th e rm o p h ile s h a r e s som e o f th e common a t t r i ­
b u te s p o s s e s s e d b y E . c o l i , i t w a s of i n t e r e s t to d e te r m in e i f th e
p r o te in s y n t h e s i z i n g m a c h in e ry o f t h i s o rg a n is m is s i m i l a r to th a t o f E .
c o li.
The p rim ary p u r p o s e o f t h i s s tu d y i s to d e te rm in e w h e t h e r a s u b c e l l u l a r p ro te in s y n t h e s i z i n g s y s te m i s o l a t e d from Tg i s c a p a b l e of
in c o r p o r a tin g 1^ C - I y s i n e u s in g p o l y a d e n y l i c a c id a s m e s s e n g e r .
The
te m p e r a tu r e d e p e n d e n c e on t h i s in c o r p o r a tio n w a s to be s t u d i e d a n d th e
9
- c o n d itio n s for maximum a c t i v i t y w e re to be d e te r m in e d .
S e d im e n ta tio n
v e l o c i t y e x p e r im e n ts w e re to be p e rfo rm ed u tiliz in g p u rif ie d rib o s o m e s to
i n d i c a t e th e s i z e o f t h e s e r i b o s o m e s .
3
(a)
MATERIALS AND METHODS
B a c te r ia l s t r a i n s : Tg , a n u n c l a s s i f i e d th e rm o p h ilic b a c t e r i a r e s e m ­
b lin g Therm us a g u a t i c u s , w a s o b ta in e d from D r. J. T erry U h lric h (21)
a s a fro z e n c u l t u r e .
T his b a c t e r i a w a s u s e d a s a s o u rc e o f rib o s o m e s
a n d e n z y m e s e m p lo y e d in t h i s s t u d y .
R ib o so m es a n d e n z y m e s from
E s c h e r i c h i a c o li MRE-600 u t i l i z e d in p a r a l l e l s t u d i e s w e re a g e n e ro u s
g ift from M r. C h a r le s A. R o e s s n e r of th e Z o o lo g y a n d E n to m o lo g y D e p a rt
m e n t, M o n ta n a S ta te U n i v e r s i t y .
(b)
B a c te r ia l g ro w th m e d ia : R eg u lar s t u d i e s e m p lo y e d a g ro w th medium
c o n ta i n in g 0.2% try p to n e (D ifco ) p lu s 0.2% y e a s t e x t r a c t (D ifco) in a
b a s a l s a l t s s o lu tio n c o n ta in in g :
0.04 g (NH4 )2 SO4 , 0.07 g KOI, 0.22 g
N a C l , 0.7 g N a g HPO4 - 7 Hg0 , 0.01 g C a(N O 3) 2 • 4H2 0 , 0.01 g M g S 0 4 '
7 H g O , a n d 5.0 m is o f H u tn e r 1s tr a c e e le m e n t s o lu tio n (23) p e r l i t e r of
sin g ly d is tille d w a te r.
The pH w a s a d j u s t e d to 8.2 w ith 0. 8 N N aO H
a n d a u t o c l a v e d for 45 m in u te s - a t 1 5 -2 0 lb s of s te a m p r e s s u r e .
(c)
I n c u b a tio n c o n d itio n s :
A R e s e a r c h S p e c i a l t i e s tu b e h e a t e r (M o d e l
# 2 1 2 8A) w a s u s e d for in c u b a t io n of u n s h a k e n tu b e c u l t u r e s .
B atch c u l t u r e s w e re grow n in a N ew B ru n sw ick G y ro to ry S h a k e r
(M o d e l #625) w h ic h w a s a d a p t e d to a c c o m m o d a te 12 2 - l i t e r c u ltu re
fla k s.
A c o n s t a n t a i r te m p e ra tu re (7 0 ° ± 2 °C ) w a s u s e d .
A g ita tio n
p ro v id e d th e o n ly m e a n s of a e r a tin g t h e s e c u ltu r e s w ith a c o n c o m m ita n t
i n c r e a s e in g e n e r a t i o n t i m e .
G row th w a s m o n ito red b y w ith d ra w in g
11
a l i q u o t s a n d d e te r m in in g th e tu r b i d ity o f 640 nm in a B a u s c h & Lomb
S p e c tr o n ic 2 0 (2 4 ).
W h e n tu r b i d ity r e a c h e d a l e v e l c o rre s p o n d in g to m id - lo g a r ith m ic
p h a s e (Ag^g o f 0.40 to 0.50) th e c u ltu r e f l a s k s w e re re m o v e d from th e
s h a k e r a n d t r a n s f e r r e d to th e c o ld room ( 4 ° C ) . A fter 1 -2 h ours of
c o o l i n g , . t h e c u l t u r e s w e re h a r v e s t e d w ith a S h a rp ie s S u p e r C e n t r i f u g e ,
Type T - I .
The c e l l s w e re re m o v e d from t h e r o t o r , r e s u s p e n d e d in an
a p p r o p r ia te b u f f e r , a n d r e c e n tr if u g e d in th e S o rv a ll RC-2 a t 7 ,0 0 0 x g ,
0 ° C , f o r '30 m i n u t e s .
The c l e a r s u p e r n a t a n t w a s d e c a n t e d a n d th e c e l l s
w e re s to r e d a t - 2 0 °C u n t i l u s e .
(d)
R ib o n u c le a s e re m o v a l: Any c o n ta m in a tin g RNAase a c t i v i t y w a s
re m o v e d b y th e fo llo w in g m e th o d s .
o v e rn ig h t b e fo re u s e .
(I) G l a s s w a r e w a s h e a t e d a t 2 4 0 °C
(2) Buffers w e re b e n to n ite t r e a t e d fo r 4 hours b y
a d d in g 2 d ro p s of a b e n t o n i t e s u s p e n s i o n .
f i I t e re d a n d s to r e d in th e c o l d .
They w e re t h e n m illi p o r e -
(3) N a lg e n e or p o ly c a r b o n a te tu b e s w e re
s o a k e d in 5% HgOg fo r 30 m in u te s a n d t h e n r in s e d w ith c o p io u s am o u n ts
o f d o u b ly d i s t i l l e d w a t e r .
(4) O th e r m a te r ia l w a s a u t o c l a v e d for 20
m in tu e s a t 15 lb s o f s te a m p r e s s u r e .
(e)
B u ffe rs : All b u ff e rs w e re p re p a re d w ith d o u b l y - d i s t i l l e d w a te r w h ic h
h a d a minimum s p e c i f i c r e s i s t a n c e o f 670 KAcm *.
This d o u b l y - d i s t i l l e d
w a t e r w a s p re p a r e d from h o u s e d i s t i l l e d w a t e r w ith a C o rn in g AG-2
d istilla tio n a p p a ra tu s .
-12
N e c e s s a r y p H a d j u s t m e n t s w e re m ad e w ith c o n s t a n t b o ilin g h y d ro ­
c h l o r i c a c i d (th e c o n s t a n t b o ilin g p o rtio n o f a d i s t i l l e d 1:2 m ixture o f a
r e a g e n t h y d r o c h lo r ic a c i d w ith d i s t i l l e d w a te r).. The pH m e a s u re m e n ts
w e re m ad e w ith a C o rn in g M o d e l 12 p H M e te r a f t e r p rio r s t a n d a r d i z a t i o n
w ith th e a p p r o p r ia te C o le m a n pH s t a n d a r d s .
All b u ff e rs w e re t r e a t e d w ith b e n t o n i t e to s e l e c t i v e l y rem ove a n y
c o n ta m in a ti n g r i b o n u c l e a s e a c t i v i t y .
Two d ro p s o f a b e n to n ite s u s p e n s i o n
w e re a d d e d to a l i t e r of b u f f e r , a llo w e d to s t a n d a t 4 ° C fo r a t l e a s t four
h o u r s , a n d t h e n re m o v e d b y f il tr a tio n th ro u g h a 50 mm M illip o re n itr o ­
c e l l u l o s e f i l t e r , in to a h e a t t r e a t e d r e a g e n t b o ttle u s in g a s t e r i l e
M illip o re s u c t i o n f i l t r a t i o n a p p a r a t u s . A fter f i l t r a t i o n , th e b u ffers w e re
s to re d a t 4 ° C .
Buffer CA
0.01 M Mg (OAc)2
0.01 M Tris
The b u f f e r w a s a d j u s t e d to pH 7.8 a t 4 0 C w ith c o n s t a n t b o ilin g H C l ,
b e n to n ite t r e a t e d for 4 h o u r s , a n d th e n f i l t e r e d .
Buffer PB
0.5 M N H 4 C l
0.01 M Mg(OAc)2
0.01 M Tris
The b u ffe r w a s a d j u s t e d to p H 7.8. a t 4 ° C w ith c o n s t a n t b o ilin g H C l ,
13
b e n t o n i t e t r e a t e d fo r 4 h o u r s , a n d th e n f i l t e r e d .
Buffer OC
0.25 M NH4 C l
0.01 M Mg(OAc)2
0.01 M Tris
The b u ff e r w a s a d j u s t e d to p H 7.8 a t 4 ° C w ith c o n s t a n t b o ilin g H C l , •
b e n to n ite t r e a t e d for 4 h o u r s , a n d t h e n f i l t e r e d .
Buffer CD
1.0 M NH4 C l
0.01 M Mg(OAc)2
0.01 M Tris
The b u ff e r w a s a d j u s t e d to pH 7.8 a t 4 ° C w ith c o n s t a n t b o ilin g H C l ,
b e n to n ite t r e a t e d for 4 h o u rs a n d th e n f i l t e r e d .
Buffer N
0.08 M N H 4 C l
0.011 M Mg(OAc)2
0 . 1 M Tris
The b u ffe r w a s a d j u s t e d to pH 8.1 a t 4 ° C (p H 7.8 a t 3 7 ° C b e c a u s e of
Tris ) w ith c o n s t a n t b o ilin g H C l , b e n to n ite t r e a t e d for 4 h o u r s , a n d th e n
filtered .
14
BC-O
.0 .1 M NH4 H C O 3
No a d j u s t m e n t w a s n e c e s s a r y ; th e p H w a s 7.88 a t room te m p e ra tu re
( 2 O0 C ).
It w a s b e n t o n i t e t r e a t e d for 4 h o u rs a n d th e n f i l t e r e d .
B C - I 0~
0.1 M N H 4 H C O 3
0.01 M Mg(OAc)2
No a d j u s t m e n t w a s n e c e s s a r y ; th e p H w a s 7.88 a t room t e m p e r a t u r e ,
(20 ° C ).
It w a s b e n t o n i t e t r e a t e d for 4 h o u rs a n d th e n f i l t e r e d .
BCz SO0.1 M N H 4 H CO3 .
0.05 M Mg(OAc)2
No a d j u s t m e n t w a s n e c e s s a r y ; th e pH w a s 7.88 a t room te m p e ra tu re
(20°).
It w a s b e n t o n i t e t r e a t e d for 4 h o u rs a n d th e n f i l t e r e d .
50-30
0 . 1 M KCl
0.01 M Tris
0.001 M M g C l2 -GH2 O
The b u ffe r w a s a d j u s t e d to pH 7.2 a t 4 ° C w ith c o n s t a n t b o ilin g H C l,
b e n to n ite t r e a t e d fo r 4 ho u rs a n d th e n f i l t e r e d .
15
pH 5'
■
0.01 M Tris
0.05 M K C l'
0.011 M Mg(OAc)2
The b u ff e r w a s a d j u s t e d to pH 5.2 a t 4 C w ith g l a c i a l a c e t i c a c i d , b e n ­
to n i t e t r e a t e d for 4 h o u r s , a n d th e n f i l t e r e d .
W h e n a s u lf h y d r y l p r o te c to r w a s d e s i r e d , 2 - m e r c a p t o e t h a n o l w a s
a d d e d to g iv e a fin a l c o n c e n tr a tio n o f 0 .0 0 6 M .
This a d d itio n to th e
b u ffe r Was m ade j u s t p rio r to u s e . ' T h e s e b u ffe rs a re d e s i g n a t e d (+) .
(f)
R e a g e n ts a n d c h e m i c a ls
DN A ase:
R ib o n u c le a s e f r e e .
O b ta in e d from W o rth in g to n B i o c h e m i c a l s .
1^ C - L y s i n e : S p e c if ic a c t i v i t y o f 318 m C i/m m o l.
P u r c h a s e d from th e
R a d io c h e m ic a l C e n t r e , A m ersham
S o lu b le RNA: C o n ta in s " s t r ip p e d " E . c o l i t - R N A .
P u r c h a s e d from
. C a l b io c h e m , c a t . # 5 5 7 1 1 2 .
S p e rm id in e : P u r c h a s e d from C a l b i o c h e m , c a t . #5 6766.
T u n g s ta te r e a g e n t (.a^):
P re p a re d from e q u a l v o lu m e m ix tu re of
and
^ 2 (see b e lo w ).
0. 6 N t r i c h l o r o a c e t i c a c i d .
A d ju s te d to pH .2 w ith s o l i d p e l l e t s of
NaOH.
CC2 :
0.02 N s o d iu m t u n g s t a t e .
a c e tic a cid .
A d ju s te d to pH 2 w ith s o lid t r i c h l o r o ­
16
(g)
R ib o so m al p r e p a r a t i o n : L a b o ra to ry m a te r ia ls a n d b u ffe rs u s e d in th is
p r e p a r a t io n w e re m ade r i b o n u c l e a s e - f r e e b y h e a t t r e a t m e n t , b e n to n ite
w a s h i n g , or e q u i v a l e n t p r o c e d u r e s .
This p ro c e d u re i s a s l i g h t m o d if ic a ­
tio n o f S t a n l e y , S a l a s , W a h b a a n d O c h o a (2 5 ).
S e v e n t y - f i v e g ram s o f fr o z e n TV, c e l l s w e re th a w e d a n d b u ffe r OA( o r N ) w a s a d d e d (I m l / g o f w e t c e l l s ) .
The r e s u l t i n g s u s p e n s i o n w a s
s t i r r e d u n t i l a c o n s i s t e n t s lu rry w a s o b t a i n e d .
(T h e s e o p e r a tio n s a n d
s u b s e q u e n t o p e r a tio n s w e re c a r rie d o u t a t Od C u n l e s s o th e r w is e s p e c i ­
fie d ).
The s lu r r y w a s p a s s e d th ro u g h a F re n c h P r e s s u r e C e l l (Aminco
o
Po w er L a b o ra to ry P r e s s ) a t a p r e s s u r e of 1 5 ,0 0 0 I b s / i n fo r 3 c o m p le te
passes.
A fter th e f i r s t p a s s , 100 /Ug of D N A ase I w e re a d d e d .
The
b ro k e n c e l l s u s p e n s i o n w a s in c u b a t e d fo r a n a d d itio n a l 30 m in u te s to
a llo w c o m p le te d i g e s t i o n o f DNA.
The c e l l l y s a t e w a s c e n tr if u g e d a t 1 8 ,0 0 0 rpm for 45 m in u te s in
th e 30 ro to r of th e S p in c o L - 2 u l t r a c e n t r i f u g e . The s u p e r n a t a n t w a s
t r a n s f e r r e d in to c l e a n , p r e t r e a t e d tu b e s a n d r e c e n tr if u g e d a t 1 8 ,0 0 0 rpm
( 3 0 ,0 0 0 xg) for 2 h o u r s .
The to p 9 0% o f S -3 0 s u p e r n a t a n t w a s t r a n s f e r r e d in to t r e a t e d 50
Ti tu b e s a n d s p u n a t 5 0 ,0 0 0 rpm ( 1 7 0 ,0 0 0 xg) for 3 h o u r s .
The top 90%
o f t h i s s u p e r n a t a n t w a s rem o v ed With a s t e r i l e s y rin g e a n d re c e n tr ifu g e d
5 0 ,0 0 0 rpm for 3 h o u r s .
The u p p e r 75% o f t h i s s u p e r n a t a n t (p u rifie d
S- IOO) w a s re m o v e d a n d d iv id e d in to 3.0 ml a l i q u o t s .
T h e s e a liq u o ts
17
w e re s t o r e d a t - 9 0 °C in th e REVCO f r e e z e r u n t i l u s e .
The p e l l e t s r e s u l t i n g from th e f i r s t 5 0 ,0 0 0 rpm c e n t r if u g a tio n w e re
c o v e r e d w ith I. 5 m is o f b u ff e r B+ a n d a llo w e d to s o l u b i l i z e o v e rn ig h t in
th e r e f r i g e r a to r w ith o c c a s i o n a l s t i r r i n g .
The tu b e s w e re th e n f il le d
w ith b u ff e r B+ a n d c e n tr if u g e d a t 2 5 ,0 0 0 rpm (Low S p e e d W ash ) in th e
50 Ti r o t o r .
The to p 90% of th e s u p e r n a t a n t w a s rem o v ed w ith a s t e r i l e s y rin g e
a n d t r a n s f e r r e d i n t o t r e a t e d tu b e s .
rpm for 4 h o u r s .
It w a s t h e n c e n tr if u g e d a t 4 0 ,0 0 0
The s u p e r n a t a n t w a s re m o v e d w ith a s t e r i l e s y rin g e
a n d th e p e l l e t s w e re c o v e r e d w ith I. 5 m is of b u ffe r O C + a n d a llo w e d to
s o l u b i l i z e o v e r n ig h t.
The r ib o s o m a l s u s p e n s i o n w a s d i l u t e d to ~ 5 0 mis w ith b u ffe r OC
a n d a p p l i e d to d ie th y la m in o e th a n e (D E A L )-c e llu lo s e co lu m n ( s e e
M a t e r i a l s a n d M e th o d s :
DEAE co lu m n p r e p a ra tio n ) w h ic h h ad b e e n
+
+
p r e v i o u s l y e q u i l i b r a t e d w ith OC . A p p ro x im a te ly I l i t e r of OC w a s
p a s s e d th ro u g h th e co lu m n a n d th e e l u a t e m o n ito re d .
The co lu m n w a s th e n e lu te d w ith I l i t e r of b u ffe r OD .
w e re c o l l e c t e d a n d th e AggQ w a s d e te r m in e d .
F ra c tio n s
The f r a c tio n s c o n ta in in g
th e g r e a t e s t a m o u n t o f Aggg m a te r ia l w e re p o o le d a n d c e n tr if u g e d a t
4 0 , 0 0 0 rpm for 4 h o u rs in th e 50 Ti r o to r .
The p e l l e t s from t h i s c e n t r i -
fu g a tio n w e re c o v e r e d w ith 1.0 mis o f b u ff e r BC-IO
s o l u b i l i z e for 4 h o u r s .
a n d a llo w e d to
The c o n c e n tr a tio n - w a s e s t i m a t e d b y Aggg
m e a s u r e m e n t s , th e r ib o s o m e s w e re b o t t l e d in 0.4 ml a l i q u o t s a n d s to r e d
18
in th e REVCO f r e e z e r a t - 9 0 ° C u n til u s e .
(h)
DEAE co lu m n p r e p a r a tio n :
(I) O n e h u n d re d gram s o f D E A E -c e llu lo se
(Biorad c e l l e x - D ) w e re s o a k e d in 2 l i t e r s o f d o u b ly d i s t i l l e d w a t e r for
o n e h o u r.
(2) The s u s p e n s i o n w a s s t i r r e d a n d l e f t to s e t t l e for 2 0 m in ­
u t e s a n d th e fin e p a r t i c l e s p o u re d o ff.
tw ic e.
This p r o c e s s w a s r e p e a t e d
(3) th e DEAE w a s w a s h e d w ith I l i t e r o f 0.1 N N a O H for 20 m in ­
u t e s a n d th e s lu r r y w a s v a cu u m f il te r e d in a B u ch n er f u n n e l.
d u re w a s r e p e a t e d t w i c e .
This p r o c e ­
(4) The DEAE w a s w a s h e d to n e u t r a l i t y w ith
w a te r and ag ain su ctio n filte re d .
(5) O ne l i t e r of 95% e t h a n o l w a s
a d d e d a n d th e s u s p e n s i o n w a s s t i r r e d a n d l e f t s ta n d i n g for 20 m in u te s .
The DEAE c e l l u l o s e w a s f il te r e d a n d t h i s p r o c e s s w a s r e p e a t e d .
(6)
E th a n o l t r a c e s w e re re m o v e d b y w a s h i n g w ith d i s t i l l e d w a t e r a n d f i l t r a ­
tio n .
(7) A s e c o n d w a s h i n g w ith I l i t e r of 0 . 1 N N aO H w a s p e rfo re d .
(8) The DEAE w a s w a s h e d to n e u t r a l i t y w ith d o u b ly d i s t i l l e d w a t e r a n d
su ctio n filte re d .
(9) The DEAE w a s r e s u s p e n d e d in th e a p p r o p r ia te
b u ffe r a n d a fin e s lu r r y w a s m a d e .
(10) The r e s i n w a s p o u re d in to a
Biorad Type D co lu m n (65 cm x 3. 5 .cm) a n d p a c k e d u n d e r 10 p s i n itr o g e n
p r e s s u r e a t room t e m p e r a t u r e .
OC
+
The co lu m n w a s w a s h e d w ith 2 lite r s of
o
a t 4 C a n d s to r e d i n th e c o ld room u n til u s e .
The f in a l d im e n s io n s
o f th e p a c k e d co lu m n w e re 45 x 3 cm .
(i)
P r e p a r a tio n of pH 5' fr a c tio n :' A 3. 0 ml a liq u o t o f th e p u rif ie d S -1 0 0
w a s th a w e d a n d a p p lie d to a G -5 0 S e p h a d e x colum n (2 cm x 12 cm)
19
e q u ip p e d w ith a n e x tr a c o a r s e p o r o s i t y s i n t e r e d g l a s s d i s c .
The pH 5'
f r a c tio n w a s p r e p a r e d b y e lu tin g w ith pH 5' b u ffe r a n d th e y e llo w is h
p r e c i p i t a t e w a s c o l l e c t e d in a 12 ml c o n i c a l c e n tr ifu g e t u b e .
The p r e ­
c i p i t a t e w a s c e n tr if u g e d a t to p s p e e d in a c l i n i c a l c e n tr if u g e for 15
m in u te s a n d th e s u p e r n a t a n t w a s d i s c a r d e d .
The p r e c i p i t a t e w a s w a s h e d
tw i c e b y r e s u s p e n s i o n in p H 5' a n d c e n t r i f u g a t i o n .
The f in a l p r e c i p i t a t e
w a s r e s u s p e n d e d in 0 .5 - 1 . 0 m is of B C -IO + a n d u s e d a s th e pH 5' fra c tio n .
(j)
In v itr o in c o r p o r a tio n p r o c e d u r e s :
I n c u b a tio n s w e re p e rfo rm ed in 12
ml r i b o n u c l e a s e - f r e e c o n i c a l c e n tr if u g e t u b e s .
G l a s s w a r e a n d c h e m i c a ls
w e re a l s o m ade r i b o n u c l e a s e fre e b y m e th o d s a l r e a d y d e s c r i b e d .
Each
tu b e c o n t a i n e d th e fo llo w in g m a t e r i a l s a n d a l l r e a g e n ts w e re m ade up in
B C -1 0 .
The s t a n d a r d in c u b a t io n v o lu m e w a s 0.5 m i s .
0.025 m is o f 0.08 M ATP
0. 02 5 m is o f 5 x 10
-4
GTP
0.025 m is of 4 m g /m l E. c o li t-RNA
I4
0.025 mis of 10 /iC i/m l 1^ C - L y s i n e
. 0.025 m is Oi 4 M S p e rm id in e
0. 0 1 0 -0 . 50 m is of 2 m g /m l p o ly A
0 .0 1 0 - 0 .5 0 m is o f Tg r ib o s o m e s (12 m g/m l)
0 .0 1 0 - 0 .5 0 mis. pH 5' fr a c tio n
The f in a l v o lu m e a n d Mg++ c o n c e n t r a t i o n w a s a d j u s t e d t o 0.5 mis. b y
a d d in g th e a p p r o p r ia te a m o u n ts o f BC-O+ a n d BC-SO+ b u f f e r s .
20
All a d d i t i o n s o f th e r e a g e n t s w e re d o n e in a n i c e b a t h .
The
r e a c t i o n w a s i n i t i a t e d b y th e a d d itio n o f th e e n e r g y s o u rc e a n d r a d i o ­
a ctiv ity .
The t u b e s w e re b r ie f ly m ix ed a n d p l a c e d in a w a t e r b a th a t
th e a p p r o p r ia te t e m p e r a t u r e .
Upon c o m p le tio n of th e i n c u b a t i o n , th e
r e a c t i o n w a s s to p p e d b y a d d in g i c e - c o l d t u n g s t a t e r e a g e n t .
(k)
C o u n tin g o f t o t a l a c i d - p r e c i p i t a b l e c o u n t s : The r e a c t i o n w a s
te r m in a te d b y t h e a d d itio n o f 3. 0 mis o f i c e - c o l d
a n d 0 . 1 mis of 10%
t r i c h l o r o a c e t i c a c i d a n d th e tu b e w a s p l a c e d in a n i c e b a t h .
F lo ccu la­
tio n w a s c o n tin u e d fo r 30 m in u te s a n d th e tu b e s w e re c e n tr if u g e d in th e
c l i n i c a l c e n tr if u g e fo r 10 m in u te s a t to p s p e e d .
The s u p e r n a t a n t w a s
a s p i r a t e d a n d th e p r e c i p i t a t e w a s d i s s o l v e d in 0.5 mis o f 0.2 N KOH
a n d l e t s t a n d for 3.0 m in u te s a t room t e m p e r a t u r e .
The s o l u t i o n w a s a c i d i f i e d b y th e a d d itio n of 3. 0 m is o f
I drop of 6 N H C l .
and
It w a s a llo w e d to s t a n d for 2 - 4 h o u rs a n d th e n
s u c t i o n f i l t e r e d th ro u g h a M illip o re f i l t e r m a n ifo ld u s i n g M illip o re n itr o
c e llu lo se f il te r s .
The e m p ty tu b e w a s r i n s e d w ith cu a n d th e r in s e w a s
a d d e d to th e f i l t e r s .
This s t e p w a s p erfo rm ed 3 t i m e s .
The f u n n e ls o f th e M illip o re m a n ifo ld w e re a l s o r i n s e d 3 tim e s
w ith a g . ' The n i t r o c e l l u l o s e f il te r s w e re p la c e d on p l a n c h e t s and d rie d
a t 7 0 °C for 15 m i n u t e s .
The s a m p l e s w e re c o u n te d for I m in u te in a
N u c l e a r - C h i c a g o M o d el 4312 3 cm w in d o w e d g a s flow c o u n te r .
21
(I)
S e d im e n ta tio n v e l o c i t y m e a s u r e m e n ts :
R ib o so m es w e re th a w e d a n d
d i l u t e d to th e a p p r o p r ia te c o n c e n tr a tio n w ith 5 0 -3 0 b u ff e r.
They w e re
d i a l y z e d a g a i n s t 2 - 5 0 0 ml c h a n g e s o f 5 0 -3 0 b u ffe r w ith th e fo rc e d flow
d ia ly z er.
A p p ro x im a te ly 0. 5 m is w e re p l a c e d in th e A N -H ro to r o f th e
B eckm an M o d e l E U l t r a c e n t r i f u g e .
A fter th e ro to r r e a c h e d s p e e d a t
3 6 , 0 0 0 rp m , p h o to g ra p h s w e re t a k e n e v e r y 4 m i n u t e s .
The g l a s s s lid e
w a s d e v e lo p e d a n d m e a s u r e m e n ts w e re m ade w ith a N ik o n M ic ro c o m ­
p arato r.
The s e d i m e n t a t i o n c o n s t a n t s w e re c a l u c l a t e d b y u s e of th e
fo llo w in g e q u a t i o n s :
/ 1\
o
_ 2 .3 0 3 x s lo p e
SOBS- ^
x
S2 0 , w
t SOBS>
4
(a)
EXPERIMENTAL RESULTS
R e q u lre m e a t o f 2 - P h o s p h o e n o lp y r u v ic a c i d (PEP) a n d p y ru v a te k i n a s e
. in
14
C - I y s i n e i n c o r p o r a t i o n : M o s t in v itr o p ro te in s y n t h e s i z i n g
s y s t e m s re q u ir e PEP a n d p y ru v a te k i n a s e a s a n ATP g e n e r a tin g s y s te m
s i n c e h ig h l e v e l s of ATP a re fo u n d to be in h ib ito r y (2 6).
p h i l i c s y s t e m s (13) do n o t h a v e th is r e q u ir e m e n t.
Some th e rm o ­
It w a s o f i n t e r e s t to
d e te r m in e w h e th e r a n ATP g e n e r a tin g s y s te m w a s o b lig a to r y for ^ C l y s i n e in c o r p o r a tio n or i f i n c r e a s e d c o n c e n t r a t i o n s of ATP c o u ld be
su b stitu ted .
(b)
The r e s u l t s of t h i s i n v e s t i g a t i o n a re sh o w n in Table 3 .
S e d im e n ta tio n p ro file of T^ r ib o s o m e s :
The s e d im e n ta tio n p ro file
o f r ib o s o m e s w a s i n v e s t i g a t e d to e s t i m a t e th e s i z e o f Tg r i b o s o m e s .
Two c o n c e n t r a t i o n s of r ib o s o m e s w e re u s e d a n d th e v a l u e s w e re c o r r e c t e d
to SgQ w - The s e d i m e n t a t i o n p ro file i s r e p r e s e n t e d in Fig u re I a n d th e
s e d i m e n t a t i o n v a l u e s a re sh o w n in T a b le 2 .
(c)
O p tim a l c o n d itio n s for in c o r p o r a tio n o f ^ C - I y s i n e u s in g p o l y a d e n y l i c a c i d a s m e s s e n g e r a t 3 7 ° , 4 5 ° , 5 5 ° , and 6 5 °C : The c o n d itio n s
n e c e s s a r y for o b ta in in g maximum in c o r p o r a tio n w e re i n v e s t i g a t e d .
T h e s e e x p e r im e n ts w e re lim ite d to th e d e te r m in a tio n of optim um c o n c e n ++
t r a t i o n s of Mg
, s p e r m i d i n e , a n d p o l y a d e n y l i c a c id r e q u ir e d for m a x i­
mum b i o s y n t h e s i s of l y s i n e p e p t i d e s .
No a tte m p t w a s m ade to i n v e s t i ­
g a te th e c o n c e n t r a t i o n o f rib o s o m e s a n d e n z y m e s n e c e s s a r y for
maximum in c o r p o r a tio n s i n c e t h e s e v a r i a b l e s w e re s t a n d a r d i z e d in e v e r y
in c o rp o ra tio n .
23
M g++ t i t r a t i o n : The c o n c e n tr a tio n o f Mg++ n e c e s s a r y for maximum
in c o r p o r a tio n o f 1^ C - I y s i n e in a n in v itro p ro te in s y n t h e s i z i n g s y s te m
w as in v e stig ate d .
The c o n c e n tr a tio n o f Mg
a p p r o p r ia te a m o u n ts o f BC-O
a n d B C -5 0
+
4-
w a s v a r ie d b y u s in g th e
b u ff e r.
T h e s e s t u d i e s w e re
p e rfo rm ed in th e p r e s e n c e a n d a b s e n c e o f s p e r m id in e .
c o n c e n t r a t i o n o f 4 mM s p e r m id in e w a s u s e d .
tin u e d for 30 m i n u t e s .
An a rb itra ry
All i n c u b a t i o n s w e re c o n ­
The r e s u l t s a t e a c h te m p e ra tu re a r e p r e s e n te d in
F ig u re s 2 - 5 a n d s u m m a riz e d in T ab le I .
S p e rm id in e t itr a ti o n :
The a r b itr a r y c o n c e n tr a tio n o f 4 mM s p e r m i-
d in e w a s u t i l i z e d in d e te r m in in g th e optim um c o n c e n tr a tio n o f Mg
r e q u ir e d for maximum i n c o r p o r a t i o n .
4 -4-
It w a s n e c e s s a r y to d e te r m in e th e
optim um c o n c e n t r a t i o n o f s p e r m id in e r e q u ir e d for maximum s y n t h e s i s of
14
C -Iy sin e p e p tid e s.
The r e s u l t s of t h e s e i n v e s t i g a t i o n s a re d e p ic t e d
in F ig u re s 6 -9 a n d s u m m a riz e d in T a b le I .
P o ly a d e n y lic a c i d titr a ti o n :
The c o n c e n tra tio n o f p o ly a d e n y l ic
a c i d r e q u ir e d for maximum b i o s y n t h e s i s w a s i n v e s t i g a t e d u s in g th e
p r e v i o u s l y d e te r m in e d optim um c o n c e n t r a t i o n s of Mg
+
4-
and sp erm id in e .
T h e s e r e s u l t s a re p r e s e n t e d in F ig u re s 1 0 -1 3 a n d s u m m a riz e d in Table I .
(d)
I n c o rp o ra tio n of ^ C - I y s i n e c o m p a re d a t 3 7 ° , 4 5 ° , 5 5 ° , a n d 65QC:
T h e s e s t u d i e s w e re perfo rm ed s im u l t a n e o u s l y a t e a c h te m p e ra tu re
u s in g th e s a m e p r e p a r a t io n of pH 5' p r e c i p i t a b l e p rp te in f r a c tio n to
p r e c lu d e th e p o s s i b i l i t y of v a r i a t i o n in e n z y m e c o n c e n t r a t i o n . All i n c u -
\
24
b a tio n s w e re perform ed fo r 30 m in u te s .
The r e s u lts o f th e s e e x p e rim e n ts
a re sh o w n in F ig u re 1 4.
Ce) Time s tu d y o f in c o rp o ra tio n o f ^ C - I y s i n e a t 3 7 ° , 4 5 ° , 5 5 ° , and 650 C
u s in g th e pH 5' p re c ip ita b le p ro te in fr a c tio n an d rib o s o m e s from T^:
The in c o rp o ra tio n o f ^ C - I y s i n e w a s fo llo w e d a s a fu n c tio n o f tim e
u s in g th e pH 5' p ro te in fra c tio n an d rib o s o m e s is o la te d from
• A liq u o ts
w e re re m o v e d e v e ry 15 m in u te s for a t o t a l in c o rp o ra tio n tim e of 120
m in u te s .
The re s u lts , o f th is in v e s t ig a ti o n a re sh o w n in F ig u re s 1 5 -1 8 .
The 15 a n d 30 m in u te v a lu e s w e re s e l e c t e d fo r c o m p a riso n to th e JE. c o li
s y s te m a n d t h e s e v a lu e s c a n b e fo u n d in F ig u re s 2 0 -2 3 .
(f)
Time s tu d y o f in c o rp o ra tio n o f ^ C - I y s i n e a t 3 7 ° , 4 5 ° , 5 5 ° , an d 6 5 °C
u s in g th e pH 5' p r e c ip ita b le p ro te in fr a c tio n an d rib o s o m e s from E . c o l i :
The in c o rp o ra tio n o f
14
C - I y s in e w a s fo llo w e d a s a fu n c tio n of tim e
u s in g th e pH 5' p r e c ip ita b le p ro te in fr a c tio n an d rib o s o m e s is o la te d from
E . c o li.
The c o n c e n tr a tio n s o f th e pH 5' p ro te in , fr a c tio n a n d rib o s o m e s
from E . c o li w e re a p p ro x im a te ly th e sa m e a s th e pH 5' p ro te in fra c tio n
a n d rib o s o m e s from Tg . The sa m e o p tim a l c o n c e n tr a tio n s of s p e r m id in e ,
p o ly a d e n y lic a c i d , an d Mg
w e re u s e d .
The to ta l in c o rp o ra tio n tim e
w a s 30 m in u te s an d a liq u o ts w e re rem o v ed e v e ry 5 m in u te s .
from th is in v e s tig a tio n a re sh o w n in F ig u re 1 9 .
The r e s u lts
The 15 a n d 30 m inute
in c o rp o ra tio n v a lu e s w e re u s e d in c o m p a ris o n w ith th o s e o b ta in e d w ith
th e Tg s y s te m an d c a n be foun d in F ig u re s 2 0 - 2 3 .
5
DISCUSSION
The u s e o f s y n th e t ic h o m o rib o p o ly n u c le o tid e s a s m e s s e n g e r RNA
in th e s tu d y o f in v itr o p ro te in s y n th e s is in E . c o li w a s in itia te d b y
N ire n b e rg a n d M a tth a e i (2 7 ).
■
T h e ir c l a s s i c e x p e rim e n ts sh o w e d th a t
p o ly u rid y lic a c id in th is in v itro s y s te m c o u ld d ir e c t th e in c o rp o ra tio n o f
14
C - p h e h y la la n in e in to a p o ly p h e n y la la n in e p e p tid e .
T his d is c o v e ry
w a s a n e le g a n t c o n firm a tio n o f th e m e s s e n g e r h y p o th e s is p ro p o s e d by
Ja c o b a n d M onod (2 8 ).
L a te r e x p e rim e n ts in v o lv in g random an d n o n -ra n d o m c o p o ly m e rs
(29) s h o w e d th a t th is in v itro s y s te m c o u ld prom ote th e in c o rp o ra tio n o f
th e re m a in in g a m in o a c i d s .
T h e se in v e s tig a tio n s w e re r e s p o n s ib le for
th e e v e n tu a l e lu c id a tio n o f th e g e n e tic c o d e .
R ec e n t e x p e rim e n ts (30)
h a v e sh o w n th a t a n in v itr o p ro te in s y n th e s iz in g s y s te m from E. c o li
c a n s y n th e s iz e a n a c tiv e en zy m e u s in g th e a p p ro p ria te c o n d itio n s .
The re q u ire m e n ts for a n in v itro p ro te in s y n th e s iz in g s y s te m h a v e
b e e n w e ll d e fin e d (3 1 ).
The c o m p le x ity of th e s y s te m d e p e n d s u p o n th e
ty p e o f m e s s e n g e r e m p lo y e d .
N a tu ra l m e s s e n g e r s re q u ire a num ber o f
fa c to rs in a d d itio n to th o s e re q u ire d w ith s y n th e tic m e s s e n g e r s .
M any o f th e s y n th e tic m e s s e n g e r s s tu d ie d in a n in v itr o s y s te m
from E . c o li p o s s e s s c o n s id e r a b le s e c o n d a ry s tru c tu re a n d a re in a c tiv e
as m essen g ers.
T h e rm o p h ilic b a c te r ia p o s s e s s h e a t - s t a b l e rib o so m e s
an d e n z y m e s w h ic h s h o u ld a llo w th e in v e s tig a tio n o f t h e s e n o rm a lly
in a c tiv e m e s s e n g e r s a t h ig h e r te m p e r a t u r e s .
T h e se h ig h e r te m p e ra tu re s
26
w o u ld c a u s e p a r tia l d e n a tu r a tio n o f th e m e s s e n g e r s , a llo w in g th e ir
a tta c h m e n t to rib o s o m e s an d th e ir s u b s e q u e n t tr a n s la t io n .
The c o d in g
p ro p e rtie s o f th e s e m e s s e n g e r s c o u ld th e n b e s tu d ie d .
The Tg in v itro s y s te m w a s in v e s tig a te d to d e te rm in e w h e th e r th is
s y s te m c o u ld be a p p lie d to th e s tu d y o f in a c tiv e m e s s e n g e r s .
P o ly -
a d e n y lic a c i d , w h ic h d o e s n o t p o s s e s s e x te n s iv e s e c o n d a ry s t r u c t u r e ,
w a s u s e d a s m e s s e n g e r to d e te rm in e th e te m p e ra tu re optim um o f th e
in v itr o s y s te m .
M o st in v itr o p ro te in s y n th e s iz in g s y s te m s re q u ire a n ATP g e n e r­
a tin g s y s te m .
H o w e v e r, F ried m an (13) h a s sh o w n th a t in c r e a s e d c o n ­
c e n tr a tio n s o f ATP a n d GTP w ill s u b s tit u te fo r th is s y s tm e in B_.
s te a r o th e r m o p h iltis .
S in c e th e P E P -p y ru v ate k in a s e ATP g e n e ra tin g
s y s te m w a s u s e d in e a r ly s tu d ie s o f
14
C - I y s in e in c o rp o ra tio n in th e
Tg s y s te m , i t s e e m e d lik e ly th a t h ig h e r te m p e ra tu re s c o u ld in a c tiv a te
th is m e s o p h ilic e n zy m e s y s te m w ith c o n s e q u e n t lo s s o f in c o rp o ra tio n .
T his p ro b lem w a s in v e s t ig a te d in th e Tg s y s te m a t 3 7 °C .' The ATP
an d GTP c o n c e n tr a tio n s w e re in c r e a s e d a n d a P E P -p y ru v ate k in a s e s y s ­
te m w a s u s e d a s a. c o n tr o l.
The r e s u lts from T ab le 3 in d ic a te th a t
tw ic e th e n o rm al c o n c e n tr a tio n o f ATP a n d GTP w ill s u b s tit u te fo r th e
ATP g e n e r a tin g s y s te m in th e TV, in v itr o s y s te m .
The s e d im e n ta tio n p ro file of th e Tg rib o s o m e s w a s in v e s tig a te d to
d e te rm in e w h e th e r t h e s e rib o s o m e s d iffe re d s ig n if ic a n tly from th o s e o f
E. c o li.
T his e s tim a tio n a t tw o c o n c e n tr a tio n s of rib o s o m e s sh o w ed
th e r e w a s no s ig n if ic a n t d iff e r e n c e (T ab le 2) w h en th e s e d im e n ta tio n .
v a lu e s w e re c o r r e c te d to Sori
.
ZUzW
The v a r ia tio n s n o te d in th e s e tw o
d e te r m in a tio n s m ay b e d u e to th e d if f e r e n c e s in c o n c e n tr a tio n .
A m a th ­
e m a tic a l e x p r e s s io n ( s e e M a te ria ls an d M ethods), p ro v id in g a p p ro x im a te
v a lu e s w a s u s e d in th e c a lc u la tio n .
C rude rib o s o m e s from Tg w e re p re p a re d b y d if f e r e n tia l c e n tr if u ­
g a tio n a n d w e re c o m p le te ly in a c tiv e in s u p p o rtin g -'-^Q -Jysin e in c o rp o ra ­
tio n .
A c c o rd in g ly , rib o s o m e s w e re p u rifie d b y D E A E -c e llu lo se c h ro m a ­
to g ra p h y (25) a n d w e re o n ly p a r tia lly a c tiv e a t 3 7 ° C . A s im ila r s itu a tio n
e x i s t s w i t h 'EL s te a ro th e rm o p h ilu s rib o s o m e s w h en p u rifie d b y th e sam e
m e th o d .
■
A lg ra n a ti a n d L e n g y e l (14) o v e rc a m e th is p ro b lem b y a d d in g
s p e r m id in e , a n a tu r a l c o m p o n e n t o f r ib o s o m e s , to th e ir in c o rp o ra tio n
m ix tu r e s .
T his p ro c e d u re w a s tr ie d w ith th e in v itr o s y s te m o f T g .
The a d d itio n of 4 mM sp e rm id in e to th e Tg s y s te m a t 3 7°C r e s u lte d
in a lo w e rin g o f th e M g
-I—I-
t o 10 mM ( s e e F ig u re 2 ) .
tio n .
c o n c e n tr a tio n from 12 mM w ith o u t sp e rm id in e
T h ere w a s a ls o a 5 -f o ld in c r e a s e of in c o rp o ra ­
E a rlie r s tu d ie s w ith o u t s p e rm id in e sh o w e d optim um in c o rp o ra tio n
a t 12 mM Mg
++
a n d a g ra d u a l d e c r e a s e o f in c o rp o ra tio n to 2 0 mM Mg
++
At 4 5 ° C , th e a d d itio n o f 4 mM sp e rm id in e r e s u lte d in a lo w e rin g
o f th e Mg
++
c o n c e n tr a tio n to 6 m M .
in c r e a s e o f .in c o r p o r a tio n .
T h ere w as a ls o a n o th e r 5 -fo ld
W ith o u t s p e r m id in e , o p tim a l in c o rp o ra tio n o f
^ C - I y s i n e o c c u rre d a t 12 m M .(se e F ig u re 3 ).
At 5 5 ° C , th e a d d itio n of 4 mM s p e rm id in e a g a in r e s u lte d in an
•
28
o p tim a l Mg
-H-
c o n c e n tra tio n o f 6 mM a n d a 6 -f o ld in c r e a s e o f in c o rp o ra ­
tio n ( s e e F ig u re 4 ).
At th is te m p e r a tu r e , th e re w a s a m ore p ro n o u n c e d
re q u ire m e n t for Mg++ in th e p r e s e n c e o f 4 mM s p e r m id in e . W ith o u t
s p e r m id in e , th e o p tim a l re q u ire m e n t o f M g++ w a s 12 mM a s p re v io u s ly
n o te d .
At 6 5 ° C , th e a d d itio n o f 4 mM s p e rm id in e r e s u lte d in a s lig h t
in c r e a s e o f th e Mg
re q u ire m e n t to 8 mM a n d a m ore p ro n o u n c e d p e a k
o f in c o rp o ra tio n ( s e e F ig u re 5 ). W ith o u t s p e r m id in e , h a rd ly a n y
14
in c o rp o ra tio n o f
C - I y s in e to o k p l a c e .
The r e a s o n fo r th e v a r ia tio n in Mg
te m p e ra tu re is u n c le a r .
++
c o n c e n tra tio n a s a fu n c tio n of
T his p h e n o m en o n m ay b e d u e to p a r tia l d e n a tu r e -
tio n o f rib o s o m a l s i t e s or h e a t- in d u c e d c o n fo rm a tio n a l c h a n g e s in th e
m e s s e n g e r w h ic h m ay a f f e c t th e b in d in g o f th is m e s s e n g e r to th e r ib o ­
so m es.
The e f f e c t o f s p e rm id in e in p ro m o tin g h ig h e r le v e l s o f in c o rp o ra tio n
is a ls o unknow n.
P o ly a m in e s , s u c h a s p u tr e s e i n e , s t a b i l i z e th e r-RNA
o f th e rm o p h ilic rib o s o m e s b u t h a v e n o e f f e c t on th e r-RNA o f E. c o l i .
S p e rm id in e , on th e o th e r h a n d , s t a b i l i z e s b o th ty p e s o f f-RNA (1.3)
u s in g th e rm a l d e n a tu r a tio n p ro f ile s a s a m e a s u re of s t a b i l i t y .
W e is s (32) a n d Kimes (33) h a v e sh o w n th a t th e a d d itio n o f s m a ll
a m o u n ts o f s p e rm id in e o r p u tr e s c in e to e x te n s iv e ly p u rifie d 30s and 50s
s u b u n ite s o f E_. c o li rib o s o m e s c a u s e s s tr u c tu r a l c h a n g e s .
T h e se
a lte r a t io n s m ake th e s u b u n it m ore s u s c e p tib le to a tta c k b y p a n c r e a tic
29
r ib o n u c le a s e an d a d e c r e a s e in s e d im e n ta tio n c o n s ta n t. .H o w ev e r, a
d e ta ile d s tu d y o f th e rm o p h ilic rib o s o m e s u s in g th e s e m e th o d s h a s no t
been d o n e .
S in c e th e u s e o f s p e rm id in e r e s u lte d in a c h a n g e of Mg"^+ c o n ­
c e n tr a tio n d e p e n d e n c e a t h ig h e r te m p e r a t u r e s , i t w a s c o n c e iv a b le th a t
h ig h e r te m p e ra tu re s m ay c h a n g e th e re q u ire m e n t fo r s p e r m id in e .
A c co rd ­
in g ly , s p e rm id in e titr a ti o n s w e re p erfo rm ed a t e a c h te m p e r a tu r e ..
At 3 7 ° C , th e le v e l o f ^ S - I y s i n e in c o rp o ra tio n w ith 10 mM Mg++
r e a c h e d a p la te a u a t 4 mM s p e rm id in e (F ig u re 6).
in c o rp o ra tio n o f
14
At 4 5 ° C , optim um
C - I y s in e w a s o b ta in e d a t 4 mM sp e rm id in e in th e
p r e s e n c e o f 6 mM Mg++ (F ig u re 7 ).
W h en th e te m p e ra tu re w a s in c r e a s e d
O
++
to 55 C w ith 6 mM Mg , th e re w a s a s lig h t in c r e a s e in th e optim um
s p e rm id in e c o n c e n tra tio n to 5 mM (F ig u re 8 ).
At 6 5 ° C , th e optim um
s p e rm id in e c o n c e n tra tio n w a s a g a in 5 mM in th e p r e s e n c e o f 8 mM Mg
-f-+
(F ig u re 9 ).
The r e a s o n for th is v a ry in g s p e rm id in e c o n c e n tra tio n is a ls o
u n c le a r .
It is te m p tin g to s p e c u la te th a t th e sp e rm id in e m ay be s u b +4.
s titu tin g for Mg
a s th e te m p e ra tu re i s in c r e a s e d b u t th e r e s u lts a t
3 7 °C do n o t s u p p o rt th is n o tio n .
If th e rib o s o m e s a re b e co m in g p a r tia lly d e n a tu re d a t h ig h e r te m p ­
e r a t u r e s , th e a m o u n t of m e s s e n g e r a b le to a tta c h m ay i n c r e a s e .
S in ce
h ig h e r te m p e ra tu re m ay a ls o c a u s e lo s s o f s e c o n d a ry s tr u c tu r e of
m e s s e n g e r , m ore m e s s e n g e r c o u ld b in d to th e rib o s o m e s an d r e s u lt in
30
a g r e a tly e n h a n c e d in c o rp o ra tio n .
A c c o rd in g ly , a p o ly a d e n y lic a c id
titr a ti o n w a s p e rfo rm ed a t e a c h te m p e r a tu r e .
At 3 7 ° C , th e am o u n t o f m e s s e n g e r re q u ire d fo r o p tim a l in c o rp o ra ­
tio n s e e m s to b e 100 jug (F ig u re 1 0 ).
a d d itio n o f th is am o u n t o f m e s s e n g e r .
The s y s te m is s a tu r a te d a f te r
At 4 5 ° C , o p tim a l in c o rp o ra tio n
o f 1^ C - I y s in e ta k e s p la c e a t 1 00 jug a n d th e s y s te m is s a tu r a te d (F ig u re
1 1 ).
W h en th e te m p e ra tu re is in c r e a s e d to SS0 C , s a tu r a tio n is a g a in
re a c h e d a t 100 /ig (F ig u re 1 2 ) . ' At SS0 C , th e s y s te m is a g a in s a tu r a te d
w ith 100 jUg (F ig u re 1 3 ).
A c c o rd in g ly , s u b s e q u e n t in v e s tig a tio n s w e re
p e rfo rm ed u s in g t h e s e c o n c e n tra tio n s o f p o ly a d e n y lic a c i d .
S in c e th e optim um c o n c e n tr a tio n s o f s p e r m id in e , Mg
+4*
, a n d p o ly ­
a d e n y lic a c id h a d b e e n d e te rm in e d a t e a c h te m p e r a tu r e , a c o m p a riso n o f
in c o rp o ra tio n s o f
14
C - I y s in e w a s in v e s t ig a te d a t th e s e te m p e ra tu re s „
T h e se s tu d ie s a tte m p te d to d e te rm in e th e o p tim a l te m p e ra tu re o f th is
in v itr o p ro te in s y n th e s iz in g s y s te m .
In c o rp o ra tio n s o f 1^ C - I y s in e w e re p erfo rm ed s im u lta n e o u s ly a t
e a c h te m p e ra tu re a n d th e sam e pH 5' p re c ip ita b le p ro te in fr a c tio n w a s
u s e d to p reclu d e, a n y v a r ia tio n in e n zy m e c o n c e n tr a tio n .
The r e s u lts of
th is in v e s tig a tio n (F ig u re 14) sh o w o p tim a l a c tiv ity a t SS0 C . The
re d u c e d in c o rp o ra tio n o f 1^ C - I y s in e a t 6 5 °C w a s n o t e x p e c te d s in c e
th e B_. s te a ro th e rm o p h ilu s s y s te m h a s e n h a n c e d a c ti v ity a t 6 5 °C (1 4 ).
H o w e v e r, in c o rp o ra tio n v a lu e s a t 6 5 °C a re a p p ro x im a te ly th e sam e a s
at37°C.
31
S in c e th e in c u b a tio n tim e in th e a b o v e s tu d ie s w a s lim ite d to 30
m in u te s , th e r a te an d e x te n t o f in c o rp o ra tio n o f
tig a te d for 120 m in u te s .
14
C - I y s in e w a s i n v e s ­
S a m p le s w e re re m o v e d e v e ry 15 m in u te s .
At
3 7 ° C , th e r a te o f in c o rp o ra tio n w a s m uch s lo w e r th a n a t 4 5 °C o r 5 5 °C
(F ig u re s 1 5 , 16 an d 17) b u t a p p ro x im a te ly th e sa m e a s a t 6 5 ° C .
H ow ­
e v e r , th e e x te n t o f in c o rp o ra tio n i s m uch g r e a te r a t 3 7 °C th a n a t 6 5 °C
(F ig u re s 15 a n d 1 8 ).
T his r e s u l t s e e m s to in d ic a te th a t d e n a tu r a tio n
o f som e c o m p o n e n t o f th e s y s te m m ay b e p ro c e e d in g a t th is h ig h te m p ­
e ra tu re .
M o st s tu d ie s in v o lv in g in v itr o p ro te in s y n th e s is in th e rm o p h ilic
s y s te m s a re c o m p a re d to th e E . c o li s y s te m .u n d er th e sa m e c o n d i t i o n s .
S im ila r c o n c e n tr a tio n s o f rib o s o m e s an d pH 5 p r e c ip ita b le p ro te in f r a c ­
tio n s from E . c o li w e re in c u b a te d a t th e v a rio u s te m p e ra tu re s u n d e r
id e n tic a l c o n d itio n s p re v io u s ly d e te rm in e d fo r T ^ .
T h e se in c u b a tio n s
w e re p erfo rm ed fo r 30 m in u te s a n d s a m p le s w e re rem o v ed e v e ry 5 m in u te s
At 3 7 ° C , th e i n i t i a l r a te o f ^
- l y s i n e in c o rp o ra tio n w ith th e Jjh
c o li s y s te m (F ig u re 19) w a s s lo w e r th a n a t 4 5 ° C a lth o u g h th e e x te n t o f
in c o rp o ra tio n w a s a p p ro x im a te ly 10% h ig h e r.
At 5 5 ° C , th e i n i t i a l r a te
o f in c o rp o ra tio n w a s m uch s lo w e r th a n a t 3 7 ° C o r 4 5 ° C .
14
The e x te n t of
C - I y s in e in c o rp o ra tio n w a s a p p ro x im a te ly 38% o f th a t o c c u rrin g a t
3 7 ° C . At 65°C , th e le v e l o f in c o rp o ra tio n w a s o n ly a few h u n d re d
c o u n ts a b o v e b a c k g ro u n d .
It w a s d e s ir a b le to co m p are b o th th e E . c o li an d Tg s y s te m s
32
d ir e c tly .
For th is c o m p a ris o n , p re v io u s d a ta w e re e x tr a c te d an d p lo tte d .
In F igure 2 0 , b o th s y s te m s a re f a irly c o m p a ra b le a t 3 7 ° C .
At 4 5 ° C , th e
s y s te m h a s a p p ro x im a te ly th e sam e i n i t i a l ra te b u t th e in c o rp o ra tio n
o f ^ C - I y s i n e c o n tin u e s to a g r e a te r e x te n t (F ig u re 2 1 ).
At 5 5 ° C , th e
Tb, s y s te m h a s a m uch g r e a te r i n i t i a l r a te (F ig u re 22) an d a p p ro x im a te ly
3 1 / 2 - f o ld a s m uch in c o rp o ra tio n o f " ^ C - I y s i n e . At 6 5 ° C , th e Tg s y s te m
h a s 20 tim e s th e am o u n t o f ^ C - I y s i n e in c o rp o ra te d a s th e E_. c o li
s y s te m (F ig u re 2 3 ).
The r e s u l t s p r e s e n te d in th is s tu d y h a v e sh o w n th a t th e in v itro
p ro te in s y n th e s iz in g s y s te m from th is e x tre m e ly th e rm o p h ilic g ram ­
n e g a tiv e b a c te r ia d o e s s u p p o rt p o ly a d e n y lic a c id d ir e c te d ^ C - I y s i n e
in c o rp o ra tio n .
A lth o u g h g ra m -n e g a tiv e th e rm o p h ilic b a c te r ia h a v e b e e n
is o la te d a n d th e rib o s o m e s s tu d ie d , no o n e h a s re p o rte d a s u c c e s s f u l
in v itr o p ro te in s y n th e s iz in g s y s te m . •
The s e d im e n ta tio n c o n s ta n ts o f th e th e rm o p h ilic rib o s o m e s a re
c o m p a ra b le to th o s e of E. c o l i .
fo r a c ti v ity a t 5 5 ° C .
T his s y s te m h a s a n o p tim a l te m p e ra tu re
The r e a s o n fo r re d u c e d in c o rp o ra tio n a t 6 5 °C is
u n c le a r s in c e i t w a s h o ped th a t th is th e rm o p h ilic s y s te m w o u ld h a v e
a h ig h e r te m p e ra tu re optim um th a n o b ta in e d w ith B_. s te a r o th e r m o p h ilu s .
TABLE I
OPTIMUM CONDITIONS for
Temperature (0C)
M g ** (XlO-3M)
IO
37
'
'
45
INCORPORATION of
■
14
C-LYSINE
Spermidine (x 10” M)
. ' ,
4
•'
Poly-ACpg)
'
IOO
6
'4
IOO
55
6 ''
5
/0 0 /
65
8
a
/0 0
.
34
. TABLE 2
.Tz -Rib o s o m a l s e d i m e n t a t i o n
SEDIM ENTATION
CONCENTRATION
"
. 2 .0 m g /m l
32,9 ± 0:6
constants
CONSTANT*
50.2 ± 0.4
1
\ 5.8 m
g / m l
-
j
: 32\ 2 ±
0;4
45 . 9 + 0.7
.
e x p r e s s e d os S y e d b e r g s
( IQJ 3s e c .)
35
TABLE 3
% 14C-LYSINE INCORPORATION
%
SY STEM
INCORPORATION
PEP
' Pyruvate
kinase
2 .0 j j M ATP
0 .0 1 2 JtM G T P
IO O
-
2 . 0 jsM A T P
.
. 63
O.OfEjuM GTP
2 .0 juM A T P
■
• '’
4 . 0 JJM A T P
IOS ■
GTP
- 102
4 . 0 JtM A T P '
0 .0 2 5 JiM
GTP
'
94
0 .0 2 5 j s M G T P
0 . 0 1 2 JJ M
1
'
'■
■
.
36
F ig u re I .
S e d im e n ta tio n p ro file o f rib o s o m e s
T2 rib o s o m e s a t th e d e s ig n a te d c o n c e n tr a tio n s w e re d ia ly z e d o v e rn ig h t
a g a in s t 5 0 -3 0 b u ffe r.
T h is s o lu tio n w a s th e n c e n trifu g e d a t 3 6 ,0 0 0
rpm in th e B eckm an' M o d e l E a n a ly t ic a l u ltr a c e n tr if u g e e q u ip p e d w ith
S c h lie re n o p t i c s .
A fte r th e ro to r a tta in e d s p e e d , p h o to g ra p h s w e re
ta k e n a t 4 m in u te i n t e r v a l s .
SEDIMENTATION VELOCITY RESULTS
[R/bosomej=2.0 mg/ml
t - 0'
t = Q1
t=4'
f = /2'
t = 16'
-
~_/vA—I
^Ribosome^ = 5.8 mg/ml
t=0'
A ___
t = 4'
Ju
t=8'
-
JL
t = /2'
:
yvl--- >
MG'
X
38
F ig u re 2 .
M g+ + titr a ti o n a t 3 7°C
In c o rp o ra tio n o f 1^ C - I y s in e w a s in v e s t ig a te d a t 3 7 °C in th e p r e s e n c e
■:>
an d a b s e n c e o f 4 mM s p e rm id in e w ith v a ry in g c o n c e n tr a tio n s o f M g+ + .
■
I nc uba t i on tim e w a s 30 m in u te s an d tr ic h lo r o a c e t ic a c id - s o d iu m tu n g s ta te
in s o lu b le r a d io a c tiv ity w a s m e a s u re d .
39
+
Spermidine
Spermidine
8
14
40
F ig u re 3 .
M g++ titr a ti o n a t 4 5 °C
In c o rp o ra tio n o f 14C - I y s in e w a s in v e s t ig a te d a t 4 5 °C in th e p r e s e n c e
an d a b s e n c e o f 4 mM s p e rm id in e w ith v a ry in g c o n c e n tr a tio n s o f M g+ + .
In c u b a tio n tim e w a s 3 0 m in u te s a n d tr i c h lo r o a c e t ic a c id - s o d iu m tu n g ­
s ta t e in s o lu b le r a d io a c tiv ity w a s m e a s u re d .
COUNTS
per
M INUTE
(xIO
+ S perm idine
— Sperm idine
[ / W g + *]
f x /O - 3 MJ
42
•
•,
.
F ig u re 4 .
M g+"1" titr a tio n a t 5 5° C
In c o rp o ra tio n o f ^ C - I y s i n e w a s in v e s t ig a te d a t 5 5 °C in th e p r e s e n c e
an d a b s e n c e o f 4 mM s p e rm id in e w ith v a ry in g c o n c e n tr a tio n s o f M g+ + .
• In c u b a tio n tim e w a s 30 m in u te s a n d tr ic h lo r o a c e tic a c id - s o d iu m tu n g ­
s t a t e in s o lu b le r a d io a c ti v ity w a s m e a s u re d .
.i. I
4- S p e r m i d i n e
COUNTS
per
M INUTE
( x IO
43
-
Sperm idine
44
I
F ig u re 5 .
M g++ titr a ti o n a t 6 5° C
In c o rp o ra tio n o f 14C - I y s in e w a s in v e s t ig a te d a t 6 5 °C in th e p r e s e n c e
an d a b s e n c e o f 4 mM s p e rm id in e w ith v a ry in g c o n c e n tr a tio n s o f M g+ + .
In c u b a tio n tim e w a s 30 m in u te s a n d tr i c h lo r o a c e t ic a c id - s o d iu m tu n g ­
s ta t e in s o lu b le r a d io a c tiv ity w a s m e a s u re d .
45
4- Spermidine
— Spermidine
46
F ig u re 6 .
S p e rm id in e titr a ti o n a t 3 7 °C
14
n
In c o rp o ra tio n o f
C - I y s in e w a s in v e s t ig a te d a t 37 C in th e p r e s e n c e o f
++
10 mM Mg
a n d v a ry in g c o n c e n tr a tio n s o f s p e r m id in e . In c u b a tio n tim e
w a s 30 m in u te s an d tr i c h lo r o a c e t ic a c id - s o d iu m tu n g s ta te in s o lu b le
r a d io a c tiv ity w a s m e a s u re d .
47
[sperm;cfme]
fx IO M)
48
F ig u re 7 .
In c o rp o ra tio n o f
o f 6 mM Mg
-I--J-
14
S p e rm id in e titr a ti o n a t 4 5 °C
o
C - I y s in e w a s in v e s t ig a te d a t 45 C in th e p r e s e n c e
a n d v a ry in g c o n c e n tr a tio n s o f s p e r m id in e . In c u b a tio n
tim e w a s 30 m in u te s a n d tr i c h lo r o a c e t ic a c id - s o d iu m tu n g s ta te
in s o lu b le r a d io a c ti v ity w a s m e a s u re d .
COUNTS
per
MINUTE
(xIO
49
[Spermi di ne^
(xlO~3 M)
I
50
•.
•/• ■
.
- \
-
F ig u re 8 .
S p e rm id in e t itr a ti o n a t .55 °C
■ 14
o
In c o rp o ra tio n o f
C - I y s in e w a s in v e s t ig a te d a t 55 C in th e p r e s e n c e o f
6 mM Mg
a n d v a ry in g c o n c e n tr a tio n s o f s p e r m id in e .
In c u b a tio n tim e
w a s 30 m in u te s .a n d tr ic h lo r o a .c e tic a c id - s o d iu m tu n g s ta te in s o lu b le
r a d io a c ti v ity w a s m e a s u re d .
51
uj
20
5,2
F ig u re 9 .
In c o rp o ra tio n o f
8 mM Mg
14
S p e rm id in e titr a ti o n ' a t 65°C
o
C - I y s in e w a s in v e s t ig a te d a t 65 C in th e p r e s e n c e o f
a n d v a ry in g c o n c e n tr a tio n s o f s p e r m id in e . In c u b a tio n tim e
w a s 30 m in u te s a n d t r i c h lo r o a c e t ic a c id - s o d iu m tu n g s ta te in s o lu b le
r a d io a c tiv ity w a s m e a s u re d .
COUNTS per MINUTE (xIO
53
[ sperm/ d/ne ]
(xIO
M)
54
F ig u re 1 0 .
In c o rp o ra tio n o f
1 0 mM Mg
14
P o ly a d e n y lic A cid T itra tio n a t 3 7 °C
o
C - I y s in e w a s in v e s t ig a te d a t 37 C in th e p r e s e n c e o f
a n d 4 mM s p e r m id in e .
a c id w a s v a r ie d a s i n d i c a t e d .
m in u te s .
m e a s u re d .
The c o n c e n tr a tio n o f p o ly a d e n y lic
In c u b a tio n s w e re c o n tin u e d fo r 30
The tr i c h lo r o a c e t ic a c id - s o d iu m tu n g s ta te r a d io a c tiv ity w a s
55
pg POLY-A
56
F ig u re 1 1 .
P o ly a d e n y lic A cid T itra tio n a t 4 5 °C
In c o rp o ra tio n o f ^ C - I y s i n e w a s in v e s t ig a te d a t 4 5 ° C in th e p r e s e n c e
6 mM M g++ a n d 4 mM s p e r m id in e .
a c id w a s v a r ie d a s i n d ic a te d .
m in u te s .
The c o n c e n tr a tio n o f p o ly a d e n y lic
I n c u b a tio n s w e re c o n tin u e d fo r 30
The tr i c h lo r o a c e t ic a c id - s o d iu m tu n g s ta te in s o lu b le r a d io ­
a c t i v i t y w a s m e a s u re d .
.......
.......................
"
' ' '
jjg POLY-A
COUNTS per MINUTE (xIO
58
I
F ig u re 1 2 .
In c o rp o ra tio n o f
14
P o ly a d e n y lic a c id titr a ti o n a t 5 5 °C
o
C - I y s in e w a s in v e s t ig a te d a t 55 C in th e p r e s e n c e o f
-f— h
6 mM Mg
a n d 5 mM s p e r m id in e .
The c o n c e n tr a tio n o f p o ly a d e n y lic
a c id w a s v a r ie d an d in c u b a tio n s w e re c o n tin u e d fo r 30 m in u te s .
The
tr i c h lo r o a c e t ic a c id - s o d iu m tu n g s ta te in s o lu b le r a d io a c ti v ity w a s
m e a su re d .
ZTE
-
■ ■
'
-
-
- ■,
COUNTS
per MINUTE (xIO
59
pg POLY-A
60
F ig u re 1 3.
In c o rp o ra tio n o f
8 mM Mg
+ -J-
14
P o ly a d e n y lic A cid titr a ti o n a t 6 5 °C
o
C - I y s in e w a s in v e s t ig a te d a t 65 C in th e p r e s e n c e o f
a n d 5 mM s p e r m id in e .
a ic d w a s v a r ie d a s in d i c a t e d .
m in u te s .
In c u b a tio n s w e re c o n tin u e d fo r 30
The tr ic h lo r o a c e t ic a c id - s o d iu m tu n g s ta te in s o lu b le r a d io ­
a c tiv ity , w a s m e a s u re d -, -
r
The c o n c e n tr a tio n o f p o ly a d e n y lic
-
..................... - —
■ -
61
pg POLY-A
62
F ig u re 1 4 .
In c o rp o ra tio n o f ^ C - I y s i n e a t v a rio u s te m p e ra tu re s .
The in c o rp o ra tio n o f
te m p e ra tu re s sh o w n .
14
C - I y s in e w a s p e rfo rm e d s im u lta n e o u s ly a t th e
O ptim um c o n c e n tr a tio n s o f Mg
, s p e r m id in e , a n d
p o ly a d e n y lic a c id p r e v io u s ly d e te rm in e d fo r e a c h te m p e ra tu re w e re u s e d .
In c u b a tio n w a s c o n tin u e d fo r 30 m in u te s a n d th e tr ic h lo r o a c e tic a c i d so d iu m tu n g s ta te - in s o lu b l e r a d io a c tiv ity - w a s m e a s u re d . - ■ - - -
63
O
x
Uj
K
3
S
i
v_
Q>
Q.
CO
K
S
3
O
O
o
CENTIG RAD E
64
F ig u re 1 5 .
Time s tu d y o f in c o rp o ra tio n o f
14
o
C - I y s in e a t 37 C
In c o rp o ra tio n o f ^ C - I y s i n e w a s fo llo w e d a t 3 7 °C u s in g 10 mM M g+ + ,
4 mM s p e rm id in e a n d 100 (J.g o f p o ly a d e n y lic a c i d .
S a m p le s w e re
rem oved e v e r y 15 m in u te s a n d th e tr i c h lo r o a c e t ic a c id - s o d iu m tu n g s ta te
in s o lu b le r a d io a c tiv ity w a s m e a s u re d .
COUNTS
per
MINUTE
(x IO
65
MINUTES at 37 C
66
F ig u re 1 6 .
Time s tu d y o f in c o rp o ra tio n o f
14
o
C - I y s in e a t 45 C
In c o rp o ra tio n o f ^ C - I y s i n e w a s fo llo w e d a t 4 5 °C u s in g 6 mM Mg"*"1",
4 mM s p e r m id in e , a n d 100 /ig o f p o ly a d e n y lic a c id .
S a m p le s w e re
rem o v ed e v e r y 15 m in u te s a n d th e tr i c h lo r o a c e t ic a c id - s o d iu m tu n g s ta te
in s o lu b le r a d io a c tiv ity w a s m e a s u re d .
67
MINUTES at 4 5 C
68
F ig u re 1 7 .
Time s tu d y o f in c o rp o ra tio n o f '''^ C -Iy s in e a t 5 5 °C
In c o rp o ra tio n o f " ^ C - I y s in e w a s fo llo w e d a t 5 5 °C u s in g 6 mM M g+ + ,
5 mM s p e r m id in e , a n d 100 jUg o f p o ly a d e n y lic a c i d .
S a m p le s w e re
rem o v ed e v e r y 15 m in u te s a n d th e tr i c h lo r o a c e t ic a c id - s o d iu m tu n g s ta te
in s o lu b le r a d io a c tiv ity w a s m e a s u re d .
69
MINUTES at 55° C
70
F ig u re 1 8 .
Time s t u d y o f in c o r p o r a tio n o f ^ C - I y s i n e a t 6 5°C
I n c o rp o ra tio n o f ^ C - I y s i n e w a s fo llo w e d a t 6 5 ° C u s in g 8 mM Mg+"1",
5 mM s p e r m i d i n e , a n d 100 jig o f p o l y a d e n y l i c a c i d .
S a m p le s w e re
rem o v ed e v e r y 15 m in u te s a n d th e t r i c h l o r o a c e t i c a c i d - s o d i u m t u n g s t a t e
in sto lu b le ra d io a c tiv ity w as m easu red .
71
MINUTES at 65 C
72
L
F ig u re 1 9 .
Time s t u d y o f in c o r p o r a tio n o f ^ C - I y s i n e u s in g
t h e E,. c o l i s y s t e m a t 3 7 ° , 4 5 ° , 5 5 ° , a n d 650 C
The i n c o r p o r a tio n of
14
C - I y s i n e u s i n g c o m p a r a b le c o n c e n t r a t i o n s of
pH 5 p r e c i p i t a b l e p r o te in f r a c t i o n a n d r ib o s o m e s from E . c o li w a s
in v e stig a te d .
The c o n c e n t r a t i o n s o f M g+ + , s p e r m i d i n e , a n d p o l y -
a d e n y l i c a c i d w e r e . i d e n t i c a l to t h o s e u s e d w ith th e Tg s y s t e m .
I n c u b a tio n s w e re c o n tin u e d fo r 30 m in u te s a n d s a m p l e s re m o v e d e v e r y
5 m in u te s.
The t r i c h l o r o a c e t i c a c i d - s o d i u m t u n g s t a t e i n s o l u b l e r a d i o ­
a ctiv ity w as m easu red .
73
eT
74
F ig u re 20.- C o m p a ris o n o f Tg a n d E. c o l i a t 3 7 °C
The r e s u l t s from th e tim e s t u d y o f b o th s y s t e m s w e re c o m p a re d u s in g
th e 15 a n d 30 m in u te v a l u e s e x t r a c t e d from th e p r e v io u s s t u d i e s (F ig u re
15 a n d F ig u re 1 9 ).
75
30
MINUTES at 37°C
76
F ig u re 21.. C o m p a ris o n o f T0 a n d E. c o l i a t 4 5 ° C
L
—
----------------
The r e s u l t s from th e tim e s t u d y o f b o th s y s t e m s w e re c o m p a re d u s i n g th e
15 a n d 30 m in u te , v a l u e s e x t r a c t e d from t h e p r e v io u s s t u d i e s (F ig u re 16
a n d F ig u re 1 9 ).
" b 'T
77
E. coIi
MINUTES at 4 5 ° C
78
F ig u re 2 2 .
C o m p a ris o n o f Tg a n d E. c o li a t 5 5 ° C
The r e s u l t s from th e tim e s t u d y .of b o th s y s t e m s w e re c o m p a re d u s i n g th e
15 a n d 30 m in u te v a l u e s e x t r a c t e d from t h e p r e v io u s s t u d i e s (F ig u re 17
a n d F ig u re 1 9 ).
79
MINUTES Qt 5 5 ° C
80
F ig u re 23.. C o m p a ris o n o f
a n d E. c o l i a t 6 5°C
The r e s u l t s from th e tim e s t u d y o f b o th .s y s te m s w e re c o m p a re d u s i n g th e
15 a n d 30 m in u te v a l u e s e x t r a c t e d from p r e v io u s s t u d i e s (F ig u re 18 a n d
Figure 1 9 ).
81
- T I ------------------TL
MINUTES at 6 5 ° C
LITERATURE CITED
1.
B rock, T. D . , S c i e n c e , 158., 1102 (1 9 6 7 ).
2.
K o ffle r, H . , B a c t . R e v i e w s , 2JL., 227 (19 57).
3.
G a n g h r a n , E. R. L . , B a c t . R e v i e w s , ll_, 189 (1 9 4 7 ).
4.
B au m an ,"A . J. a n d P . G . S im m o n d s , 'J. B a c t e r i o l . , 9 8 , 528 (1 9 6 9 ).
5.
D a r o n , H . H . , J . B a c t e r i o l . , 1 0 1 , 145 (1 9 7 0 ).
6.
B ro c k , T. D . a n d H . F r e e z e , J. B a c t e r i o l . , 9_8, 389 (1 9 6 9 ).
7.
M a r m u r, J . a n d P. D o ty , J. M o l. B i o l . , 5., 109 (1 9 6 2 ).
8.
M a n n in g , G . B. a n d L. L. C a m p b e ll, J . B io l. C h e m . , 2 3 6 , 2952
(1 9 6 1 ).
9.
M a n n in g , G . B . , L. L. C a m p b e ll a n d R. J . F o s t e r , J . B iol. C h e m . ,
2 3 6 , 2958 (1 9 6 1 ).
10.
C a m p b e ll, L. L. a n d G . B. M a n n in g , J . B io l. C h e m . , 2 3 6 , 2962
(1 9 6 1 ) .
11.
A m e lu x in , R. E . , B io c h im . B i o p h y s . A c ta , 1 3 9 , 24 (1 9 6 7 ).
12.
A r e a , M . , C . C a l v e r i , L. F ro n ta li a n d G . T e c c e , B io c h im . B i o p h y s .
A c t a , 87., 440 (1 9 6 4 ).
13.
F r ie d m a n , S . M . a n d I . B. W e i n s t e i n , B io c h im . B i o p h y s . A c ta , 1 1 4 ,
593 (1 9 6 6 ).
14.
A l g r a n a t i , I . D . a n d P. L e n g y e l , J . B iol. C h e m . , 241 , 1778 (1 9 6 6 ).
15.
F r ie d m a n , S . M . , R. Axel a n d I . B. W e i n s t e i n , J . B a c t e r i o l . , 9 3 ,
1521 (1 9 6 7 ).
16.
P a c e , B. a n d L. L. C a m p b e ll, P r o c . N a t ' l . A c a d . S c i . , U . S . , 5 2 ,
330 (1 9 6 7 ).
17.
B ro c k , T. D . a n d K. L. B o y le n , A p p l. M ic r o b io l. , 25_, 72 (1 9 7 3 ).
18.
Z e i k u s , J. G . , M . W . T ay lo r a n d T. D . B ro c k , B io c h im . B io p h y s .
A c ta , 204., 512 (1 9 7 0 ).
83
19.
S t e l l w a g e n , E . , M . H. C ro n lu n d a n d L. D. B a r n e s , B io c h e m is try ,
1 2 ( 8 ) , 1552 (1 9 7 3 ).
20.
F r e e z e , H. a n d T. D . B ro c k , J . B a c t e r i o l . , 1 0 1 , 541 (1 9 7 0 ).
21.
U h l r i c h , J . T . , G . A. M c F e te rs a n d K. L. T e m p le , J . B a c t e r i o l . ,
H O , 691 (1 9 7 2 ).
22.
G i l b e r t , W . a n d B. M u l l e r - H i l l , The L a c to s e O p e r o n , J . R. B ec k w ith
a n d D . Z i p s e r (e d s . ) , C o ld Spring H arb o r L a b o r a to r y , C o ld Spring
H a rb o r, N . Y. ( 1 9 7 0 ) .
23.
H u t n e r , S. H . , J . B a c t e r i o l . , 52_, 213 (1 9 4 6 ).
24.
U h l r i c h , J. T . , P h . D . T h e s i s , M o n ta n a S ta te U n i v e r s i t y (19 72).
25.
S t a n l e y , W . M . , M . S a l a s , A. J . W a h b a a n d S . O c h o a , P r o c . N a t ' I .
Acad. S c i . , U . W . , 56, 290 (1966).
26.
Z a m e n c n i k , P . C . a n d E. B. K e lle r , J . B iol. C h e m . , 2 09 , 337 (1954).
27.
N i r e n b e r g , M . W . a n d J . H . M a t t h a e i , P ro c. N a t ' l . A c a d . S c i . ,
U . S . , 47., 1588 (1 9 6 1 ).
28.
J a c o b , F. a n d J. M o n o d , J . M o l. B i o l . , 3 , 318 (1 9 6 1 ).
29.
S p e y e r , J . F . , P. L e n g y e l , C . B a s ilio a n d S. O c h o a , P ro c. N a t 'l .
A c a d . S c i . , U . S . , 4 8 , 441 (1 9 6 2 ).
30.
S c h w e i g e r , M . a n d L. M . G o ld , P r o c . N a t ' l . A c a d . S c i . , U . S . ,
63., 1351 (1 9 6 9 ).
31.
G a r d n e r , R. S . , A. J. W a h b a , C . B a s i l i c , R. S . M i l l e r , P. L en g y el
a n d J. F . S p e y e r , P ro c . N a t ' l . A c a d . S c i . , U . S . , 48., 2087 (1 9 6 2 ).
32.
W e i s s , R. L. a n d D . R. M o r r i s , B io c h e m is try , 1 2 ( 3 ) , 435 (1 9 7 3 ).
33.
K im e s , B. W . a n d D . R. M orris , B io c h e m is try , 1 2 ( 3 ) , 442 (19 73).
4
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