Download Protein Synthesis and Its Control

212.
.PROTEIN SYNTHESIS AND I T S CONTROI
D.
CALIFORNIA
J.
McCONNELL
INSTITUTE O F TECHNOLOGY
Recent advances i n molecular biology have given us a c l e a r p i c t u r e of
t h e mechanism of p r o t e i n s y n t h e s i s . The primary r o l e of DNA has been e s t a b l i s h e d as a c a r r i e r of t h e information which determines t h e l i n e a r sequence of
amino a c i d s i n t h e p r o t e i n molecules. Thus for each p r o t e i n t h e r e i s a l e n g t h
of DNA, defined i n o l d e r terminology a s a gene, or more r e c e n t l y as a c i s t r o n ,
which d e s c r i b e s t h e order of amino a c i d s f o r a s i n g l e p r o t e i n , or more
accurately , polypeptide. The g e n e t i c code i s t h e d i c t i o n a r y for t r a n s l a t i n g
t h e language of DNA s t r u c t u r e i n t o t h a t of p r o t e i n s t r u c t u r e , and t h e process
of c a r r y i n g out " t r a n s l a t i o n " i s what w i l l concern u s today.
F i r s t of all, l e t me de sc r ibe t h e s t r u c t u r e of DNA i n a l i t t l e more
d e t a i l . DNA i s a v ery long double-stranded molecule composed of f o u r subu n i t s , known g e n e r a l l y by t h e i r s u b s c r i p t s A, C, T, G, standing f o r adenylic
acid, c y t i d y l i c acid, thymidylic a c id and guanylic a c i d . Each subunit has one
phosphate and one deoxyribose r e s i d u e and e i t h e r a pyrimidine or purine
d r i v a t i v e , known as t h e base. The bases can p a i r with one another by hydrogen
bonding, so t h a t a base on one s t r a n d of t h e double-stranded DNA chemically
and p h y s i c a l l y f i t s or matches a complementary base on t h e o t h e r s t r a n d . There
a r e r e s t r i c t i o n s on t h e b a s e - p a i r s which can be formed imposed by t h e s t e r e o chemistry. Thus A can o n ly p a i r with T, and C can o n l y p a i r with G . Thi s
means t h a t once a sequence of ba se s i s l a i d down on one str a nd, t h e sequence
on t h e o t h e r s t r a n d i s p r e c i s e l y de f ine d by t h e rule of pe r missible ba se-p a i r s .
When a DNA molecule r e p l i c a t e s as a t c e l l d i v i s i o n , t h e pa r e nt double-strand
s e p a r a t e s and two n e w s t r a n d s are l a i d down on t h e pa r e nt str a nds, which a c t
as templates, t h e i r bases guiding t h e order of t h e new bases on t h e daughter
s t r a n d s . (See F ig u re 1.) The p a t t e r n of ba se s i s maintained a f t e r c e l l d i v i s i o n and each daughter c e l l r e c e i v e s an i d e n t i c a l s e t of primary information.
The DNA molecules d i v i d e and r e p l i c a t e when t h e c e l l d i v i d e s and p e r f e c t
r e p l i c a s of t h e p a r e n t molecules go t o each daughter c e l l . T h i s t h e n i s t h e
e x q u i s i t e l y b e a u t i f d chemical b a s i s of h e r e d i t y .
The DNA can be regarded as a g i g a n t i c computer t a p e , each p o s i t i o n
along i t s l e n g t h having one of f o u r p o s s i b l e b i t s of information i n t h e shape
of t h e four bases. A human c e l l c o n t a i n s approximately 170centimetres of
DNA, which r e p r e s e n t s .5 b i l l i o n b i t s sinc e t h e r e a r e 3.4 A between basep a i r s . The DNA of a b a c t e r i a l v i r u s such as T4, o r of a bacterium such as
E s c h e r i c h i a c o l i , i s found as a single molecule, while i n highe r organisms t h e
number of such molecules p e r genome i s not known.
DNA i s metabolica.lly i n a c t i v e . It p l a y s no major s t r u c t u r a l or
enzymatic r o l e s . I t s s p e c i a l i z e d f u n c t i o n i s t o c o n t a i n information; it i s a
memory. T o be u s e f u l t h e information must be t r a n s l a t e d i n t o p r o t e i n s t r u c t u r e ,
p r o t e i n s being t h e work h o r s e s of t h e c e l l determining most of i t s shape and
s t r u c t u r e ; what p r o t e i n s do not a c t u a l l y c a r r y out themselves, t h e y c o n t r o l
and f a c i l i t a t e by t h e i r c a t d y t i c a c t i v i t y .
The g e n e t i c code relates t h e sequence of bases i n DNA t o t h e sequence
Since t h e r e are only 4 bases i n DNA, and 20 comon
of amino a c i d s i n p r o t e i n .
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amino a c i d s i n p r o t e i n s , it i s c l e a r l y not a 1:1r e l a t i o n s h i p . I n f a c t it
i s a t r i p l e t code. There a r e 64 p o s s i b l e t r i p l e t s , and all except t h r e e
have been shown t o code f o r one or o t h e r of t h e amino a c i d s . Each t r i p l e t
i s c a l l e d a codon. A l l amino a c i d s have at l e a s t two codons and some, for
example l e u c i n e , have as many as six. Some examples of t h e code are given
i n Figur e 2 .
The code i s u n iv ersal, t h a t i s , it i s t h e same i n t h e most p r i m i t i v e
and t h e most evolved organisms. (Some of t h e e a r l y evidence on t h e na tur e of
t h e code came from s t u d i e s on v i r u s e s , while some came from work on human
hemoglobins.)
It i s a nonoverlapping code, so t h a t a given base does not
c o n t r i b u t e information t o ad jacent amino a c i d s . It has no "commas," so t h a t
it i s v i t a l t o start reading t h e code a t one end, and i n t r i p l e t s . It does
have "periodstr which a r e t h o s e t h r e e codons f o r which t h e r e i s no corresponding amino acid: T-A-A, T-A-G, T-G-A.
There i s a l s o a s p e c i a l f'start" codon,
T-A-G, which codes f o r a v ery d i s t i n c t i v e amino acid, N-formyl methionine,
which i s found a t t h e N-terminal end of almost all t h e p r o t e i n s i n E . c o l i .
We a r e now t h e r e f o r e ab le t o d e f i n e a gene i n chemical terms. It
i s a l e n g t h of DNA, one end of which h a s a "start" codon, and t h e o t h e r a
''stop" codon. It c o r r e s p o d s t o a p r o t e i n , and t h e sequence of ba se s of t h e
DNA i s s a i d t o be c o l i n e a r w ith t h e sequence of amino a c i d s of t h e p r o t e i n .
The hypothesis proposed a long time ago by Beadle and Tatwn of one gene-one
p r o t e i n i s t r u e i n all b u t a few s p e c i a l c a s e s .
The s t o r y i s not as simple, however. There a r e two s t r a n d s i n a
l e n g t h of DNA. We now know t h a t one of t h e s e , c a l l e d t h e rrsense" s t r a n d i s
read, while t h e o t h e r c a l l e d " m t i - s e n s e " i s not; we do not know why.
Secondly, t h e DNA i s not d i r e c t l y t r a n s l a t e d i n t o p r o t e i n s t r u c t u r e ; t h i s
process i s mediated by a secondary template, "messenger r i b o n u c l e i c acid"
(m-RNA) which i s a copy of t h e information of one s t r a n d of t h e DNA. It
migr ates from t h e nucleus t o t h e cytoplasm where t r a n s l a t i o n occurs and
p r o t e i n s a r e sy n th esized .
m-RNA i s similar t o a s i n g l e s t r a n d of DNA except t h a t each subu n i t c o n t a i n s r i b o s e as i t s sugar, and u r i d y l i c a c i d r e p l a c e s thymidylic
acid. The g e n e t i c code i s u s u a l l y w r i t t e n i n terms of m-RNA s t r u c t u r e , hence
U-A-G i n s t e a d of T-A-G. The RNA language i s so similar t o t h e DNA language
t h a t t h e pr o c ess of copying t h e l a t t e r i n t o t h e former i s c a l l e d " t r a n s c r i p tion."
The change from RNA language t o p r o t e i n language, " t r a n s l a t i o n , ) ' i s
consider ably more complicated. There i s no c l e a r stereochemical r e l a t i o n s h i p
between &no a c i d s and b a s e s or t r i p l e t s of bases. Crick proposed t h a t an
adaptor molecule w a s r e q u i r e d t o r e l a t e them t o each o t h e r and soon a f t e r wards it w a s dZscovered, i n f a c t a whole f a mily of molecules w a s i s o l a t e d ,
probably one t o r e l a t e each amino a c i d t o one or o t h e r of i t s corresponding
codons. Leucine has at l e a s t f i v e and probably six, one f o r each of i t s
codons. To confuse t h e "lay" reader, n a t u r e has determined t h a t t h e adaptor
molecule i s a l s o r i b o n u c l e i c acid of a s p e c i a l kind c a l l e d ' ' t r a n s f e r RNA" o r
'%-RNA."
Each t-RNA molecule i s c o v a l e n t l y l i n k e d at one end t o a s p e c i f i c
amino acid. I n t h e sequence of b ase s i n t h e t-RNA t h e r e i s a t r i p l e t which i s
complementary t o one of t h e codons which code f o r t h e s p e c i f i c amino a c id- t h i s t r i p l e t i s c a l l e d t h e anti-codon.
214.
W
e can now co n sid er t h e s t e p s of t r a n s l a t i o n i n more d e t a i l , and l e t
us t a k e as our model a t r i p e p t i d e N-formyl methionyl-alanyl-tyrosine. A
p o s s i b l e code for it i s shown i n Figur e 3 and would be t h e s t r u c t u r e of i t s
m-RNA. T h r e e molecules of t-RNA are r e quir e d, each w i t h i t s a ppr opr ia te a n t i codon and attach ed amino acid--N-formyl methionyl t-RNA, a l a n y l t-RNA and
g l y c y l t-RNA.
The "start" codon A-U-G, and t h e ''start" amino a c id N-formyl
methionine attach ed t o i t s t - R N A which "recognizes" t h e codon, a r e aligned;
s i m i l a r l y t h e second amino acid and i t s codon G-C-U.
The amino a c i d s t h e n
react t o form a p ep tid e bond gi ving N-formyl methionyl a la nine and d i s p l a c i n g
t h e t-RNA molecule formerly joined t o t h e N-formyl methionine. Now two amino
a c i d s are joined i n sequence t o t h e T-RNA which or igina Lly o n l y c a r r i e d t h e
a l a n i n e . The t h i r d amino a c i d ( w ith i t s t-RNA a tta c he d) i s now a ligne d
opposite i t s codon U-A-C, and a similar displacement occurs giving N-formylmethionyl a l a n y l g l y c y l t-RNA.
F i n a l l y t h i s s o l e remaining t-RNA r e sidue i s
removed and t h e p ep tid e i s complete. I n vivo t h i s f i n d s t e p occurs when
one of t h e "stop" codons i s reached.
T h is whole series of r e a c t i o n s i s c a r r i e d out by a s u b c e l l u l a r
o r g a n e l l e c a l l e d t h e ribosome. It can be viewed as a complex enzyme system.
The m-RNA molecule i s o r i e n t e d by t h e ribosome r e l a t i v e t o t h e incoming amino
a c i d s a ttach ed t o t h e i r t-RNA molecules, and components of t h e ribosome c a t a l y s e
t h e formation of t h e p ep tid e bond. It appears t h a t t h e m-RNA, which can be
very long (u p t o 10,000 bases, and maybe more), i s f e d through t h e ribosome,
each codon being t r a n s l a t e d i n sequence. Se ve r a l ribosomes can "follow" one
another a l o n g t h e m-RNA, each one making a single polypeptide--such a complex
i s c a l l e d a polysome o r polyribosome.
The s t r u c t u r e of a ribosome i s becoming better understood. It has
two subunits, each co n tain in g one molecule of "ribosomal RNA" and up t o 40
d i f f e r e n t p r o t e i n s . The role of t h i s spe c ie s of RNA i s not understood but i s
probably mainly s t r u c t u r a l , t h e c a t a l y s i s being c a r r i e d out by some of t h e
p r o t e i n s . It i s p o s s i b l e t h a t t h e r-RNA a i d s i n binding e i t h e r t h e t-RNA o r
t h e m-RNA or both. The su b u n it s of t h e ribosome are known by t h e i r sedimentat i o n c o e f f i c i e n t s 50s and 30S, as are t h e molecules of r - R U which t h e y c o n t a i n
(which are d i f f e r e n t from each o t h e r ) , 28s and 18s.
B r i e f l y t o co n sid er t h e c o n t r o l of p r o t e i n s y n t h e s i s it i s obvious
t h a t t h e r e are many components involved, and t o inf lue nc e one of them c m
s i g n i f i c a n t l y d t e r t h e whole process. I n g e n e r a l t h e following i t e m s are
needed f o r t h e s y n t h e s i s of any p r o t e i n : ( a ) a c t i v e r i b o s o r e s ; ( b ) amino
a cid s; ( c ) t-RNA; (a) t h e enzyme systems f o r charging t h e t-RNA molecules with
t h e i r r e s p e c t i v e amino a c i d s . For a s p e c i f i c p r o t e i n t o be synthesized a
s p e c i f i c m-RNA molecule must be p r e s e n t which i n t u r n means t h a t a t some time
( f a i r l y r e c e n t l y s i n c e m-RNA i s unsta ble ) t h e p a r t i c u l a r sequence of DNA must
have been t r a n s c r i b e d . Jacob md Monod have demonstrated an e l e g a n t c o n t r o l
system i n b a c t e r i a which s e l e c t i v e l y s h u t s down some sequences i n t h e DNA
while l e a v i n g o t h e r s a v a i l a b l e ; a d i f f e r e n t system has been found i n highe r
organisms by Bonner. I n both c a se s p r o t e i n molecules known as r e p r e s s o r s block
c e r t a i n r e g i o n s of t h e DNA by complexing with them.
For t h o s e who a r e i n t e r e s t e d i n t h e f i e l d I recommend the r e c e n t
book by J. D. Watson, "The Molecular Biology of t h e Gene," and one by J. Bonner,
"The Molecular Biology of Development. "
215.
Figur e 1. R e p l i c a t i o n of DNA.
-t -g- a- t -t-c -
-A-C
-T-A-A-G-
-T-G-A-T-T-C
-A-C-T-A-A-G-
-+
-A-C -T-A-A-G-
-T-T -A-T -T -C -
-+
-T -G -A-T -T -C
-
Parent double strand
Strand s e p a r a t i o n
-
-a-c -t-a-a-g
Copying
Lower-case l e t t e r s used t o denote daughter s t r a n d s .
Figure 2 .
C - C - G
proline
C - C - A
proline
A - C - A
thr e onine
A - A - A
lysine
Figur e 3.
TTT
A - U - G - G - C - U - U - A - C
N-f ormyl
methionine
alanine
glyc ine
m-RNA molecule
t -RNA molecules
with a tta c he d
amino a c i d s
216.
McCONNFZL:
Paper I n .
PEARSON:
I w a n t t o thank M r . McConnell f o r h:is c o n t r i b u t i o n t h i s
morning. I ' m Sure w e were d l g r e a t l y enlightened. We w i l l now continue with
our program by having a d i s c u s s i o n on t h e "Biosynthesis of Muscle" which w i l l
be presented by D r . C . R. Ashmore of t h e U n i v e r s i t y of C a l i f o r n i a a t Davlis.
D r . Ashmore i s i n t h e Department of A n i m a l Science. D r . Ashmore.
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