.PROTEIN SYNTHESIS AND I T S CONTROI D.

Transcription

1 212..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.

2 213. 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.

3 214. 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. "

4 215. 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

5 216. McCONNFZL: Paper In. PEARSON: I want to thank Mr. McConnell for h:is contribution this morning. I'm Sure we were dl greatly enlightened. We w i l l now continue with our program by having a discussion on the "Biosynthesis of Muscle" which will be presented by Dr. C. R. Ashmore of the University of California at Davlis. Dr. Ashmore is in the Department of Animal Science. Dr. Ashmore. ############