Revue

BIOCHIMIE, 1979, 61, 323-342.

Participation of tRNA in regulation of protein biosynthesis at the translational level in eukaryotes. L. A. O STERMAN.

(18-10-1978).

Regulation of p r o t e i n b i o s y n t h e s i s .at the translational level is believed to play an i m p o r t a n t role in b r i n g i n g about relatively r a p i d changes of cellular m e t a b o l i s m i n e~karyotes w h e n the conditions of their n u t r i t i o n a n d r e s p i r a t i o n are changed, and in h i g h e r ,organisms, in response to outer signal of humo,r,al a n d n e r v o u s origin. T r a n s c r i p t i o n processes b,ein.g located i n the n u c l e u s are less accessible to outer effects. These prooesses are involved ~in the f u n d a m e n t a l r e a r r a n gements of metabolism, e.g. u n d e r the action of g o n a d o t r o p i e hormorms. The central and p o l y f u n c t i o n a l role of tRNA in the m e c h a n i s m of t r a n s l a t i o n makes it a probable .candidate for regulating of this m e c h a n i s m . This a s s u m p t i o n is s u b s l a n t i a t e d by the following First, tRNA molecules c o n t a i n a large percentage of modified () nucleoside.s w h i c h increases from pro.karyotes to yeasts a n d f u r t h e r on to higher organisms. Second, the p o p u l a t i o n of physic.ally detectable tI~NAs not only reflect the a b u n d a n c e (>) of the genetic code but n o t i c e a b l y exceed it. F o r every tRNAs of any origin, s,everal fractions incorp o r a t i n g the same a m i n o acid into a p r o t e i n can be separated by chr~omatographic techniques. The n u m b e r of isoaccepting tRNAs is often higher than that of the c o r r e s p o n d i n g codons of the genetic code. Obviously, the m u l t i p l i c i t y of tRNAs a n d the m e c h a n i s m using ~it are genetically dete'rmined. Ho'wever, a r a t h e r long route of of tRN~A p r e c u r s o r s separates the synthesis of the respective t r a n s c r i p t s in the n u c l e u s from their utilization in the t r a n s l a t i o n apparatus. This route can be facilitated for some types of tRNAs, and h i n d e r e d for anothers, d e p e n d i n g o n the requirements ,of the cell and some outer effects. As a result, a certain prop(~rtion ~vill be established b e t w e e n the c o n t e n t of active tRNA molecules in the cell. If the ratios b e t w e e n the rates of synthesis of various proteins d e p e n d on such proportions, then p r o t e i n synthesis can be regulated

Institate of Molecular Biology, USSR Academy of Sciences, Moscow, Vavilov str. 32.

by c h a n g i n g the composition of the tRNA pool in the cell ; this, is also true for isoaccepting tRNAs. More ~over we shall try to show that the selection of already m a t u r e tRN'A molecules may take place for this purpose. The regulatory f u n c t i o n of tRN:A mol.ecules was first postulated by Ames and H a r t m a n [1] i n 1963 as a >. The hypothes,is is based on d e g e n e r a t i o n of the genetic code. Apparently, sorrm of the isoaceepting tRNAs of a certain coding specifmity m a y be r a t h e r scanty in the cellular pool of tRNAs (> tRNA structure. Eventually, they m a y be absent there, b u t do not appepar in any other not cogn,at.e positions [70]. Unders t a n d i n g of the physiological ro.le of modified nucl,eos,ides is only i~n its c h i l d h o o d so far. Ho~vever, t h e r e already is some g r o u n d to believe that this role can. be dual. On the 'one hand, modified nucleosides p r o b a b l y confe,r to all tl~NAs some c o m m o n s t r u c t u r a l peculi'arities essential ~o t h e i r f u n c t i o n ; on the other, modified nucleosides may b r i n g a b o u t s t r u c t u r a l differences necessary to open a p o s s i b i l i t y .of choice, e.g. b e t w e e n isoaccepling tl~NAs, w h i c h is p a r t i c u l a r l y i n t e r e s t i n g for the p r o b l e m of regulation. F r o m this points of view, let us analyse the .r,esu]ts of some r e c e n t investigations. I. We shall begin w i t h ribothymidine (T) whose role is the best k n o w n . The as,sumption that the ) oligonucleotide T~)CG oarries out the c o n n e c t i o n b e t w e e n tRNA and the 50S ribosoma.1 s u b u n i t t h r o u g h the c o m p l e m e n t a r y tetranucleotide CGAA :of the 5S R~NA is already well docum e n t e d [71-73]. The connecti.on is p r e c e d e d by the c o n f o r m a t i o n a l r e a r r a n g e m e n t of a tRNA molecule to o p e n the Tq)CG ,region that is poorly accessible in a free tRNA molecule. This r e a r r a n g e m e n t is i n d u c e d by the c o d o n - a n t i c o d o n i n t e r a c t i o n of tRNA a n d mRNA i a the presence of GTP a n d elongation factors [74-76]. T h e c o n t a c t of Tq)CG fixes the aminoacyl-tR,NA at the A site of the transferase centre of ,a ribosome. However, the absence of the U ~ T :or U ~ q) m o d i f i c a t i o n s does not interfere w i t h this a t t a c h m e n t but p r o b a b l y decreases its efficiency [77]. All e u k a r y o t i c i n i t i a t o r tRNAs bear the tetrarmcleotide UACG instead of Tq)CA) [78-80]. The prim a r y s t r u c t u r e of the i n i t i a t o r tR_~A.Met of mouse m y e l o m a has some ~addition.al u n c o m m o n features : a) it c o n t a i n s C instead of U at the p o s i t i o n following the antic'orlon from the 5'-end ; b) there is the m i n o r m2G :in place of the c o m m o n m ] G in the b e n d b e t w e e n the D loop (where t h e r e is no D) and the anticodo,a loop. T h e s e p e c u l i a r i t i e s are

BIOCHIMIE, 1979,

61, n ° 3.

327

p r o b a b l y in some way cormected with the initiatory function. Meanwhile, it has been f o u n d recently that A substitutes for T i~n the tRNA Ala of silkw o r m larva [82] a n d T is substituted by a modified nucleoside of a yet u n k n o w n structure in the tRNiAAsh of r a t liver [83]. Some i n f o r m a t i o n has a p p e a r e d w i t h i n the last years p o i n t i n g out that the U --> T modification in the n o n , i n i t i a t o r tRNAs of euka.ryotes is not as universal and obligatory as it was first thought [84!. It t u r n e d out that, on the average, the content of T in tissues of higher organisms is about 0.5 moles per mole of La total tRNA p r e p a r a t i o n [85]. There is no r i b o t h y m i d i n e at all i n tRNA vai of rabb:it liver, m o u s e m y e l o m a and placenta [65, 86, 87]. By the way, the complete p r i m a r y structures of these three t ~ A T M ,are n e a r l y the same. However, the .comparison of tRNA I'1'e from placenta, calf liver .and .rabbit liver has reveal.ed that the levels of their U -+ T modi'fi/catio.n varies, w i t h i n the range, of 50-70 p e r oent though all the rem, ain i n g p r i m a r y s t r u c t u r e is i d e n t i c a l [88]. Reszelbach et al [89] have found, in total beef tRNA preparations, some tRN.As w h i c h are not completely methy~ated at the same positio,n, m a i n l y tR'NLAVa] and tRNAThr. The p r o p o r t i o n s of isoaccepto.r peaks in their profiles are not the same for different itssues and change w i t h the d e v e l o p m e n t of an animal from foetal to m a t u r e stage. This m a y be c o m p a r e d w i t h changes in the r i b o t h y m i d i n e content in total tRNA of Dictyostelium discoidenm in the course of differentiation [90]. Some data are available to the effect that tRNAG~y from wheat embryo, deficient in T, even d h u i n i s h e s its t r a n s f e r activity b,eing methytated by methyl~ase from E. colt [91]. For IRNA TM, on the contrary, the rate and the level of p o l y p h e n y l a l a n i n e synthesis on the poly (U) template directly d e p e n d on the completeness of the U --> T modification [92]. Thus, it seems that in some cases the completeness of the U --> T modification may serve as a factor of the compet'itiwe selection betwc,en differ e n t fractions of tR.NA o~¢ing to a more or less effective i n t e r a c t i o n w i t h the 50S ribosomal subu n i t at the A site. I,t w o u l d be relevant to note that an E. colt s t r a i n deficient :in r i b o t h y m i d i n e may grow n o r m a l l y but ~s competitively excluded from the m i x t u r e w i t h a w i l d s t r a i n [931.

2. Modi[ied nucleosides adjacent to anticodon at its 3'-end. The data about the c o n t e n t and d i s t r i b u t i o n of these nucleosides in all tRNAs sequenced up to M~arch 1977 are compLl.ed :in a table by McGloskey and N i s h i m u r a [37]. It can be seen that in the

L. A. O s t e r m a n .

328

m a j o r i t y of tRNAs the nucleoside adjacent to an a n t i e o d o n tRNAs is modified. Excepti'o.ns may by fou.nd for valine, alani~ne, glyci,n.e and a r g i n i n e tRNAs (the la.tter w i t h CGX codons) ; let us note that codons for all these tRNAs form the fourtimes degenerated . In all other cases, a strict r e g u l a r i t y i'n, the d i s t r i b u t i o n of modified nucleosides is obvious as well as its direct correlation w i t h the base located in the first position of a codon. The modifications are relatively simple (nPG, m2A, m6A a n d mlI) for the c o d o n s b e g i n n i n g w i t h G~and G. Ho'wever, if codons begin with U, there are i n t r i c a t e l y modi'fiJed h y d r o p h o h i c derivatives of a d e n i n e (i6A, ms2i6A) o,r .even a more c u m b e r some and also h y d r o p h o b i c m i n o r yW and its derivatives adjacent to ~nticodons in tRNAs. The only e x c e p t i o n know,n so far is tRN~AT~'~ of rat liver .and silkgland where the h y d r o p h i l i c but no less .oomplex xninor t6A is looated at the same place [94]. T h e latter and its m~ethylated derivative (mt6A) are obligatorily adjacent to a n t i c o d o n s in ,all tR.NAs whose cogna.te codons begin with A. Those regularities are obviously quite significant. This m a y be illustrated by the C --> U a n d (3 --> A m u t a t i o n s in the t h i r d letter of the tRNAO~y a n t i c o d o n in E. colt (codons b e g i n n i n g w i t h G). These m:issens supressor m u t a t i o n s are i m m e d i a tely followed by modifications of the a d e n i n e adjacent to the a n t i c o d o n in tRNAOly. In the first case, t6A appears in accorda,nce w i t h the first letter of the codon AGA b e i n g supressed, and in the second, the modification yields ms2i6A (supression of the codons UGv ) [95]. The completeness of modification of the n u c l e o s i d e adjacent to an anticodon clearly influenees the capacity of tRNA to b i n d to a ribosome i,n the presence of mR'NA !69]. Meanwhile, the rate of am.i,noacylation and the ability of tRNA to complex w i t h factor Tu and GTP do~es not d e p e n d on the completeness of this modification [96]. However, as :in the case of r i b o t h y m i d i n e , data are available to the effect that the i n c o m p l e t e n e s s of modification of the nueleos,ide a d j a c e n t to a n anticodon~ m a y be n o r m a l for some tRNAs. I n d e e d it was found for a v a r i e t y of a n i m a l tissues in Littauer's l a b o r a t o r y [97, 98] that from 4 to 8 per cent of tRNA T M lack the complexly modified o y W (peroxy Y). This d,efi~iency reaches. 90 per cent for two mouse n e u r o b l a s t o m a s , and may be comp a r e d w i t h the above m e n t i o n e d data o n changes in the isoacceptor profiles of tR,N,APh~ in cancerogenesis. Let us also m e n t i o n a change in the level of the modification i~A ~ ms2i6A in the course of

BIOCHIMIE,

1 9 7 9 , 61, n ° 3.

development of Bac. subtilis in 1975 the e n z y m e activity i6A --+ A i n tRNA molecules rial and liver homogenates

[99]. M c L e n n a n found for the demodification i n ext.ract from bacte[ll}0j.

All the above evidence suggests the i m p o r t a n c e of modificatiorL of the n u c l e o s i d e adjacent to an a n t i c o d o n for the biological activity of ttLNA. At the same time, n o t h i n g is yet k n o w n about the m e c h a n i s m of these modified nucleosides i n v o l v e d in tRNA f u n c t i o n i n g . Recently F e l d m a n proposed an elegant but yet p u r e l y speculative hypothesis dealing w i t h the p r o b l e m [1011. He believes that thee ,key role i.n all the tranMatory sequence of events in a .ribosome is played by the i n t e r a c t i o n of the n,ucleoside adjacent to an a n t i c o d o n w i t h mRNA. The possibility of creating t e m p o r a r y covalent b o n d s b e t w e e n m e t h y l a t e d nucleosides and the c o m p o n e n t s of a ~ribosome t h r o u g h m e t h y l e n e bridges looks r a t h e r well argurnerLted in F e l d m a n ' s hypothesis a n d is most i n t e r e s t i n g for our f u r t h e r reasoni~ng. Let us postpone to the next section the examin a t i o n of the f u n c t i o n 'of modified nucleosides freq u e n t l y located i n the first position of an antieodon a n d p r o c e e d to other groups of minors.

3. Modified nucleosides in the loops of the secondary structure of tRNA. These are : mlA i n the T tpCG loop, D a n d Gm in the D loop, mTG, m3C a n d D in the extra loop. All of them are located on the outer side of the angle of the L - c o n f i g u r a t i , o n in the spatial model of tRNA. l,n F e l d m a n ' s hypotlmsis, they are s.upposed to p a r t i c i p a t e in c r e a t i n g the second covalent (methylene) b o n d w i t h a 50S ribosomal subunit. It is postulated that, along w i t h the above mentioned b o n d b e t w e e n the nucIeoside adjacent to an a n t i c o d o n and mRN,A, these make an axis of rotation for peptidyl-tRNA. The r o t a t i o n b r i n g s together the p e p t i d y l r e s i d u e and the amino acid of aminoacyl-tRNA. By e o m p a N n g the p r i m a r y s t r u c t u r e s of three i s o a c c e p t i n g tRNA set of rat liver, Rogg at al. [67] came to the c o n c l u s i o n that the amin.o acid specificitY of IRNA is governed by its D loop composition. H o w e v e r i n the light of later investigation [102] this c o n c l u s i o n does not look universal.

4. Methylated nucleosides from the central part of the tRNA molecule (mlG, m2G, m ~G, msC, Um etc). These minors, i n contrast to all others, are f o u n d o,nly i n tRN.As of eukaryotes. It w o u l d be

t R N A in regulation of bios{lnthesis. logical to s u p p o s e that in e u k a r y o t e s s o m e n e w a n d sp~ecitilc f u n c t i o n is c o u p l e d w i t h them, p r o b a b l y the f u n c t i o n of .regulating p r o t e i n synthesis. In the s p a t i a l m o d e l of t l ~ A , all these m i n o r s are lo.eated close to the in,her s u r f a c e of the L - - c o n f i g u r a t i o n of t h e molecule. It is at this s u r f a c e t h a t the i n t e r a c t i o n of tRNA a n d s y n t h e t a s e s tmk,es p l a c e [103~. One mino,r of this group, n a m e l y m2G, has been s h o w n to p a r t i c i p a t e in ensu.ring the m a x i m a l r a t e of tRN~A a m i n o a c y l a t i o n [104]. The role of o t h e r m i n o r s is u n c l e a r . H o w e v e r , it is i m p o r t a n t to note that all of t h e m are a l w a y s f o u n d at the same sites of the tRNA s e c o n d a r y s t r u c t u r e and in ,a n u m b e r of d,ifferent c o m b i n a tions [70]. W e m a y s u p p o s e along w i t h F e l d m a n ' s t h e o r y t h a t these mien.ors m a k e t e m p o r a r y b a n d s t h r o u g h m e t h y l e n e b r i d g e s , w i t h some c o m p o n e n t s of the t r a n s l a t i o n a p p a r a t u s . A l t e r n a t i v e l y , w e can confine to a statemen,t that some s p a t i a l distrib.ution of h y d r o p h o b i c m e t h y l groops is c r e a t e d . Ultimately, the obvious a s s e r t i o n that a c e r t a i n s p a t i a l configuration of the c e n t r a l r e g i o n of the tRNA molecule c o r r e s p o n d s to e a c h c o m b i n a t i o n of m i n o r s is also sufficient for s u p p o s i n g that ,a finite q u a n t i t y of these combin,ations dictates a final set of tRNA c o n f o r m a t i o n s w h i c h m a y be utilized for d i s c r i m i n a t i n g and selecting different tRNA groups of s i m i l a r configurations b y the t r a n s l a t o r y a p p a r a t u s . In o t h e r w o r d s , the final set of m i n o r combinations~ in the tRNA c e n t r a l p a r t m a y s e r v e as a sort of c i p h e r in a d d i t i o n to the t r a d i t i o n a l c o d o n - an.ticodon r e c o g n i t i o n . A not rarelY e n c o u n t e r e d l o c a t i o n of p s e u d o u r i dine at one end of t h e a n t i c o d o n stem or the D stem i n f l u e n c e s u n d o u b t e d l y the tRNA c o n f o r m a r i o n and, i,n this w a y , m a y also p a r t i c i p a t e ,in the configurational selection of tRNIA. T h e meani.ng of all these a s s u m p t i o n s w i l l be d i s c u s s e d later.

5. Processing o[ tRNA precursors w a s investigated v e r y i n t e n s i v e l y d u r i n g the last years, p a r t i c u l a r l y for p r o k a r y o t e s w h i l e the i n f o r m a t i o n about the m e c h a n i s m of p r o c e s s i n g in e u k a r y o t e s is m u c h less c o m p l e t e and detailed. H o w e v e r , it is a l r e a d y ,known that h e r e as well the m o d i f i c a t i o n of tRNA p r e c u r s o r also p r o c e e d s step b y step. Munns el al. [105, 106] h,ave showcn in ¢ chase )>e x p e r i m e n t s w i t h KB cell culture t h a t f o r m a t i o n of m76 is a c h i e v e d ~ i t h i n 3,0 minutes, of m l G a n d m ] G w i t h i n hour, a n d that about 3. h o u r s are n e c e s s a r y for the full compFletian of m~G, Cm a n d G,n. P a r t of the p r o c e s s i n g e n z y m e s are b o u n d to r i b o s o m e s and m a y be w a s h e d out b y a salt solution [107]. Meanwhile, G a r b e r et al. [108] r e c e n t l y BIOCHIMIE, 1979, 61, n o 3.

329

d i s c o v e r e d , i n s i l k w o r m larva, tRNA p.recursors still b e a r i n g t r i p h o s p h a t e at t h e i r ~5'- e n d but a l r e a d y 70 to 80 p e r cent modified. T h e i,nhibition of m e t h y l a t i o n b y a d d i t i o n of c y c l o l e u c i n e (a p o t e n t i n h i b i t o r of S-adenosylm e t h i o n i n e f o r m a t i o n ) into a n u t r i e n t m e d i u m d e c r e a s e s tRNA s y n t h e s i s two-fold a n d deoelerates tRNA i n c o r p o r a t i o n i n t < ) r i b o s o m e s [1091. This m a y be a t t r i b u t e d to the e x i s t e n c e of s p e c i a l nucleases b o u n d to r i b o s o m e s a n d d e s t r o y i n g tRNA p r e c u r s o r s but not m a t u r e tR*N~A molecules Ell0]. I n t e r e s t i n g d a t a ,are available on the p r e c u r s o r m o d i f i c a t i o n b e i n g d e l a y e d in the .course of r a p i d g r o w t h 'of SV-40 t r a n s f o r m e d mouse cells in culture at low c o n c e n t r a t i o n [111]. C o n t r a r y wise, Ouelette s: T a y l o r [112] have f o u n d that the pre-tRNA/tR~N~A e q u i l i b r i u m in h e p a t o m a is disp l a s e d t o w a r d s the tRNA .as c o m p a r e d to ,normal liver. This m e a n s an i n c r e a s e of the p r o c e s s i n g rate in c a n c e r o u s tissues and c o r r e l a t e s w i t h a h i g h e r a c t i v i t y of m e t h y l a s e s irL t h e m [113]. Temp o r a l .relations b e t w e e n the b e g i n n i n g of intense m e t h y l i n c o r p o r a t i o n and the changes in p r o t e i n s y n t h e s i s in r e s p o n c e to h o r m o n a l stimulation [114] i n d i c a t e a s t a t i o n a r y .excess of pre-tRNA w h o s e p r o c e s s i n g is a c c e l e r a t e d u n d e r hormon,al action. N u m e r o u s ,examples of c o r r e l a t i o n s betw e e n changes in the m o d i f y i n g e n z y m e s activities and c e l l u l a r m e t a b o l i s m m a y be found in the r e v i e w of K e r r and Borek [113]. It is e s s e n t i a l to note h.ere that, owi.ng to discon~ n e c t i o n and time s e p a r a t i o n ,of different m o d i f y i n g reactions, situations m a y arise w h e r e the r e l a t i o n s b e t w e e n the r a t e s of pro~es.sing of different, pre-tRNAs in p a r t i c u l a r i s o a c e e p t i n g tRNAs, change as a result of some. a l t e r a t i o n in the a c t i v i t y of a modi, fying enzyme, for e x a m p l e , u n d e r t h e influence of an exogen, ous metabolite. Differences in the l a b i l i t y and the t u r n o v e r rate of the modifyi,ng e n z y m e s m a y he i l l u s t r a t e d by the data of K,itchingman & F o u r n i e r [115]. T h e y s h o w e d that l e u c i n e .starvation of an a u x o t r o p h i c E. colt s t r a i n affects the modi:fi~ation of two differ e n t tRNAs but only in some, though the same, i d e n t i c a l l y l o c a t e d minors. Thus. if the p r o p o r t i o n s .of i s o a c c e p t i n g tRNAs in the cell m a y i n d e e d influence the r a t i o betw e e n the rates of bi,osynthesis of different p r o teins, the origin of changes in these p r o p o r t i o n s must b e l o o k e d for ,in the m e c h a n i s m s i n v o l v e d in the enzyme regulation of p r o c e s s i n g of tRLN'A precursors.

Codon-anticodon recognition. The u n a m b i g u i t y of codon r e c o g n i t i o n by m o d u l a t o r y tR'NA and th.e d e g e n e r a c y of the genetic

330

L. A. O s t e r m a n .

code play the c e n t r a l role in th,e m o d u l a t o r y hypothesis of Ames a H a r t m a n as well as i n the modification of this h y p o t h e s i s w h i c h will be p r o p o s e d 1.ater. Yet, the field of a p p l i c a t i o n of tbese two factors is c o n s i d e r a b l y n a r r o w e d by the of Crick [116] according to w h i c h only a n t i c o d o n s b e g i n i n g w i t h A or C m a y ensure the strict un,ambiguity of recognition. Mean~vhile, a careful study of all the p r i m a r y s t r u c t u r e s of tRNA sequenced so far shows no a n t i c o d o n s b e g i n n i n g w i t h A. A~enine is always substituted fo.r i n o s i n e c a p a b l e of interacting w i t h U, C a n d A. Up to now, however, i n o s i n e w a s f o u n d m a i n l y in the a n t i c o d o n s of tRNAs specific for a m i n o acids w i t h the four-time degenerated code. The next expansion, of possibilities for an u n a c c u r a t e recogni.tion is due to 5-methoxy-uridine (mosU) a n d uridine~5-hydroxyacetic acid (V) f o u n d i n the first position of an a n t i c o d o n in val,ine, serine a n d .alanine tRNAs though to date only for p r o k a r y o t e s [37]. These m i n o r bases m a y pair 'with U, A and G. Ultimately there are some data suggesting that C as the first letter of an a n t i c o d o n does not always ensure the u n a m b i guity of recognition. Indeed, Holmes et al. have s h o w n that tRN~A1L~u ( a n t i c o d o n CAG) is capable of s u b s t i t u t i n g for t R N A ~ u (anticodon GAG in a m u t a n t of E. colt devoid of tRNA~,~u [117]. Mitra has d e m o n s t r a t e d the .capacity of yeast tRNAL."~ ( a n t i c o d o n CUU) to recognize the c o d o n AAA [118]. This means, an ex~gansion of to C-U, C-C and C-A or the oossibility of c o d i n g w i t h only th,e tWO first bases of a codon. Jank et al. have found, i n e x p e r i m e n t s on tRNA b i n d i n g w i t h ribosomes in the presence of tri1~tets that tR~NIATM from r a b b i t liver (antic o d o n IAC) recognizes all four valirr~ codons and GUG even better than the three others [119]. However, Mitra et al. [120] do not c o n s i d e r these e x p e r i m e n t s as representative. They used three fairly well purified tRNAV,~ w i t h different anticodons (UAC, GAC and IAC). All of them displayed the classical specificity of triplet r e c o g n i t i o n in the e x p e r i m e n t s on tRNA birrding. Meanwhile, each of them p r o v e d .capable of recognizing equally well all four valine codons in the cell-free synthesis of phage MS-2 coat p r o t e i n . These data made Lagerkvist [121] propose a n,ew concept fo.r r~co~nition w h i c h is based on the possibility of some a m i n o acids to be coded for by the two first bases of a codon. Lagerkvist has postulated this possibility m a i n l y for the codon.s h a v i n g G and C in the first two positions w h e r e they form two strong bonds with

BIOCHIMIE, 1 9 7 9 ,

61, n ° 3.

anticodons. The p a i r i n g in the t h i r d position of a codon c a n n o t lead to a n y m i s c o d i n g for the cognate a m i n o acids (Pro, Gly, Ala, Arg) since all four codons form in each case a i.e. code for the same a m i n o acid. The competition based o n the c o m p l e m e n t a r y p a i r i n g in the t h i r d position of a codon t u r n s out to be a decisive factor in selecting the tRNA if there are two weak b o n d s of the A-U type in the two first positions. II ~an be seen from the code table that w i t h this type of coding in no case will four codons w i t h i d e n t i c a l two first bases code for the same a m i n o acid. This means i n a d m i s s i bility of w r o n g p a i r i n g in the t h i r d position of a codon. F o u r out of eight cases i n the i n t e r m e d i a t e type of p a i r i n g (A-U and G-C) from c o d i n g families ( i n c l u d i n g a valine family) are located i n the left half of the code table. In. contrast, all codons of the i n t e r m e d i a t e type .in the r i g h t half of the table do not e n t e r coding families, i.e. r e q u i r e the correct r e c o g n i t i o n i n the t h i r d position of a codon. It is i m p o r t a n t to realize that the regulatory f u n c t i o n s of tRNAs are to be looked for p r i m a r i l y in the cases of stringent coding, i.e. for a m i n o acids whose codons do not form coding familes. These are all the codons i n the t h i r d c o l u m n of the code table, codons for Dhenylalanine, isoleucine, cysteine a n d two codons for leucine, serine and a r g i n i n e not b e l o n g i n g to a family. Let us e x a m i n e w h e r e we stand w i t h these codons. We have to bear in m i n d as well that, w i t h i n the scope of the m o d u l a t o r y hyDothesis, the u n a m b i g u i t y of codon r e c o g n i t i o n m e a n s e l i m i n a t i o n of the w o b b l e - t y p e violations of s t r i n g e n t b a s e - p a i r i n g comDlementary in the t h i r d position of a codon (the first position of an antlcodon). Many tRNAs c a r r y modified nucleosides in the first position of t h e i r anticodo.n. Besides the above m e n t i o n e d m i n o r s tbat increase the d e g e n e r a c y of the a m i n o acid coding, there are also some others whose aclion is just the reverse. F o r instance s2U a n d all its more complicated derivatives m a y p a i r w i t h A but not w i t h G. The same is characteristic of m e t h y l ester of 5-carboxymethyl u r i d i n e mcmsU [122-124]. The deazaguanosine derivatives (Q, m a n Q a n d gal Q) pair s.ignificantly better "with U then w i t h C. A table s u m m a r i z i n g the distributi,on of modified nucleosides l.ocated in the first positions of a n t i c o d o n s a m o n g IRNAs of k n o w n p r i m a r y structures has heen .published [37]. As can he seen from lids table, m i n o r s .ensuring the u n a m b i g u i t y of codon r e c o g n i t i o n are confined exclusiveIy to this very group of tRNAs w h i c h earlier was distinguished

t R N A in regulation of biosynthesis. as a m o r e p r o b a b l e one for p e r f o r m i n g the regul a t o r y functions, a n d p a r t i c u l a r l y often to the t h i r d column~ of the code table. In some cases, the r a t i o b e t w e e n the rates of b i o s y n t h e s i s for different p r o t e i n s i n the cell m a y he regul, ated not b y a low c o n c e n t r a t i o n of m o d u l a t o r y tRNFAs, as p o s t u l a t e d b y Ames & Hartman, but, on the c o n t r a r y , b y z n i n c r e a s e d content of some i s o a c c e p t i n g tRNAs r e c o g n i z i n g only one codon. Here, the r a t e of t r a n s l a t i o n of mRNAs in w h i c h the cognate a m i n o acids are c o d e d for predomin, antely w i t h these selected codons w i l l be higher. Then, even the six-time d e g e n e r a t i o n of the genetic code for teucine, s e r i n e and arginine m a y p a r t i c i p a t e in the r e g u l a t o r y m e c h a n i s m . F o r these a m i n o .acids an i s o a c c e p t i n g tRNA w h i c h r e c o g n i z e d only one of the two d e t a c h e d codons m a y p u s h aside the tRNAs r e c o g n i z i n g the other five codo,ns. I,n this c o n n e c t i o n the r e p o r t of W e i s s e n b a c h ~ D i r h e i m e r [124] is n o t e w o r t h y . T h e y f o u n d that over 50 p e r cent of all tRNAA~g in y e a s t is r e p r e s e n t e d b y tRNAzArg w h i c h recognizes o n l y the c o d o n AGA, a n d t h a t the c o n t e n t of s i m i l a r l y special:ized t R ' N A ~ u is 2/3 of all tR'N~ALe~. The content of mLnors c a p a b l e .of .eliminating w o b l e - d e g e n e r a t i o n i n tR'N.A ,is d e t e r m i n e d b y an e n z y m a t i c regulation, p r o b a b l y c o n n e c t e d w i t h the f u n c t i o n a l state of the cell. In fact, W o n g el al. [125] have f o u n d a d i r e c t c o r r e l a t i o n b e t w e e n the rate of g r o w t h (and d e d i f f e r e n t i a t i o n ) of some Morris h e p a t o m a s and the level of i n h i b i t i o n of s u l f o t r a n s f e r a s e p r o d u c i n g the m i n o r s2U 5n them. An enzyme c a t a l y z i n g t h e i n c o r p o r a t i ' o n of guanine into m a t u r e tRNA molecules and o n l y into those h a v i n g Q in t h e i r .anticodon was r e c e n t l y f o u n d [126, 127]. It is also p o s s i b l e t h a t t h e r e a r e two e n z y m e s : first o n e i n v o l v e d in the substitution Q ~ G thus e l i m i n a t i n g the: s t r i n g e n t select i v i l y of coding, 'and another, one w h i c h pa.rticipates in the m u l t i s t a g e synthesis of Q [128]. To summ.arize, w e m a y c o n c l u d e that the p r e sence :of m i n o r s c a p a b l e of supressi, ng the c o d i n g a m b i g u i t y in the first p o s i t i o n s of a n t i c o d o n s m a y ensure, in spite of t h e w o b l e - h y p 0 t h e s i s , a stringent s e l e c t i v i t y of c o d o n r e c o g n i t i o n w h i c h is n e c e s s a r y for t h e cormept of tRN~As being i n v o l v e d in the r e g u l a t i o n of prote~n synthesis. The u n i q u e d i s t r i b u t i o n of these m i n o r s in strict c o i n c i d e n c e w i t h a w e a k e n e d t y p e of p a i r i n g for the two o t h e r bases of an a n t i c o d o n selects 1.3, a m i n o a c i d s whose i n c o r p o r a t i c m i n t o p r o t e i n m a y be used as a tool for such a r e g u l a t i o n . F o r illustration, let us discuss some e x p e r i m e n t s in w h i c h the s t r i c t selectivity of c o d o n r e c o -

BIOCHIMIE, 1979, 61, n ° 3.

331

gnition or the p r e f e r e n t i a l specificity of tRNA was ment~.oned. Hilse s~ Rudloff [129] s e p a r a t e d tRNAGly f r o m r a b b i t l i v e r i,nto two fractions. One bindLng to ribosom'es only in the p r e s e n c e of t h e t r i p l e t CAA, and the other o n l y in the p r e s e n c e of CAG. The s e c o n d f r a c t i o n m e r e l y i n c o r p o r a t e s g l u t a m i n e in the lysates of r e t i c u l o c y t e s . All four glutam~ines in the globin mRN'A are c o d e d for in f~act o n l y CAG [130]: It h a d been s h o w n e a r l i e r in the s a m e l a b o r a t o r y [131] that tRNAWl f r o m r a b b i t ,reticulocytes b i n d s m u c h better to r i b o somes in the p r e s e n c e of the t r i p l e t GUG th~n in the p r e s e n c e of the t h r e e o t h e r triplets. This select i v i t y m a y be c o m p a r e d to the fact that 1~2 out of 18 valines of r a b b i t :~-globin are c o d e d for b y GUG [130]. Valine is not a m o n g the 13 selected ¢ r e g u l a t o r y • .amino acids. H o w e v e r , this d i s p l a c e m e n t of the c o d i n g specificity of tRTffAT M in favour of the c o d o n p r e f e r e n t i a l l y u t i l i z e d in mRNA must p r o m o t e the rate of globin synthesis, i.e. p l a y s the r o l e .of a r e g u l a t o r y f a c t o r in the above mention'ed a d d i t i o n a l sense of t h i s notion. It is also possible that s t r i n g e n t s l e c t i v i t y of c o d o n r e c o g n i t i o n for two i s o a c c e p t i n g tRNAS~r of Drosophila f o u n d by W h i t e et al. [132] has the same meaning. One of t h e m r e c o g n i z e d o n l y the c o d o n UCG an the other Clearly p r e f e r r e d UCU. Let us m e n t i o n t h a t Katze f o u n d that tRNAs specific for T y r , H4s, Ash and Asp (codons N,AcV) a r e e n r i c h e d w i t h i s o a c c e p t i n g f r a c t i o n s ,containing the m i n o r s Q .and X as a result of a m e l i o r a t i o n of n u t r i e n t m e d i a for SV-40 t r a n s f o r m e d r o u t i n e cells b y a d d i t i o n of foetal calf s e r u m [133]. It is possiMe that c u l t i v a t i o n of cells in r i c h m e d i a results in a h i g h e r c o d i n g s e l e c t i v i t y w h i c h ensures the adequate r e g u l a t i o n of the p r o p o r t i o n s b e t w e e n the rates of p r o t e i n synthesis.

Amino acid coding tables. The c o n c e p t of tRNA b e i n g i n v o l v e d in the r e g u l a t i o n of p r o t e i n synthesis i m p l i e s a non-statistic c h a r a c t e r of utilization of c o d o n s for at least some amino a c i d s in m R N A s of the r e s p e c t i v e p r o t e i n s . By the time this r e v i e w was b e i n g w r i t t e n , the f o l l o w i n g struct u r a l genes w e r e comDletely s e q u e n c e d : r a b b i t ~-globin [130], o v a l b u m i n [134], t h r e e h o r m o n e s [135-1371, virus SV-40 [138], a n d two b a c t e r i o phages, ~,X-174 [139] and MS-2 [140]. T h e non-statistic c h a r a c t e r of c o d o n u t i l i z a t i o n can be demons t r a t e d in all t h e cas~es. F o r instance, only one of six c o d o n s ( C U G ) c o d e s for 16 among 18 leucines in I~-globin. In the gene for mo.use p r o i n s n l i n , all four t y r o s i n e s are e n c o d e d b y the same c o d o n . T h e t r i p l e t GAG codes for 13 among 14 m o l e c u l e s of a s p a r t i c acid in the eerie for c h o r i o n i c somatom a m m o t r o p i n . One and the same codon codes for 12 among 13 glutamins in the mRNA for r a t

L. A. O s t e r m a n .

332

growth hormone. In the SV-40 genome, 22 among 23 p h e n y l a l a n i n e molecules are e n c o d e d by the triplet UUU, etc. C o m p a r i s o n of the a m i n o acid coding tables reveals c e r t a i n regul,arities i n t h e utilization of codons. These regularities may be the same for different genes, .or the c o d i n g systems m a y vary sharply, e.g. for v i r a l proteins a n d polypeptides of a n i m a l origin. As can be seen from Table I, G is p r e f e r r e d over A in the t h i r d position of a codon in mammals. This is most p r o n o u n c e d for codons i n the ~rst c o l u m n of the coding table (NU A) and, to a less degree, for codons i n the t h i r d c o l u m n (NAA°). Note that both codon groups are characterized by a weak A-U b o n d in the second positiin. In contrast, codons termin, ated w i t h G are not used a m o n g codons i n the second c o l u m n of the table for o v a l b u m i n and SV-40. C is preferred over U i n the t h i r d position of a codon for the iv¢o h o r m o n e s ~vhereas the reverse is true f o r the two D N A - c o n t a i n i n g viruses. NAA b e i n g used in preferen,ce to NAG in the virus SV-40 may favour its gro~vth in anim.al cells. Let us discuss some other properties of this virus whi.ch are not reflected i n the table. Among 178 codons of the v i r u s vchich are located in the first c o l u m n of the coding table, only five end w i t h C. Two codons of the type AGAo are definitely p r e f e r r e d among codons for arginine. They are used 32 t i m e s while four codons .of the type C~N code for a r g i n i n e only three times. It is n o t e w o r t h y that a s i m i l a r p a t t e r n is observed for the coding of a r g i n i n e in the mR'NA for o v a l b u m i n (1'3 and 2, times). The reverse is t y p i c a l of the v i r u s ¢X-174 genome. Here codons of the type AG ~ code for a r g i n i n e 7 times whereas those of the t y p e CGN are used 81 times. In all m a m m a l i a n mRN'As, the codons CUC and CUG are p r e f e r r e d in ,coding for leucine, GAC in c o d i n g for aspartic acid, and AAG in c o d i n g for lysine. In the S¥-40 gen,onle however, l e u c i n e is e n c o d e d preferenti.ally by the codons UUA a n d UUG, l y s i n e by the codon AAA, etc. I t is too early to i n t e r p r e t these regularities in physiological terms,. However, we must emphasize that the selection of codons b e i n g used is definitely non-statistical i n a n n m b e r of Cases. The example given below deals w i t h the growth of a bacteriophage. Nevertheless; its discussion w o u l d be befitting since it presents by n o w the only possibility to qualitatively compare the k n o w n ratio b e t w e e n the rates of synthesis of fun.ctionally related p r o t e i n s a n d the c h a r a c t e r of their coding. F i e r s et al. [140-142] completely seq u e n c e d the RNA of the b a c t e r i o p h a g e MS-2. This

BIOCHIMIE, 1 9 7 9 ,

61, n ° 3.

RNA codes for three p r o t e i n s : the infective protein A, the phage coat protein, and replicase. There are i n i t i a t o r codons a n d adjacent to them, nontranslated i n i t i a t o r regions of the genome, for each of these proteins. Therefore, each p r o t e i n can be synthesized i n d e p e n d e n t l y from the two others. These .proteins are c o m p a r a b l e in s.ize. Y'et, there are 180 copies of the ,coat p r o t e i n per molecule of p r o t e i n A in a m a t u r e phage particle. This means that the gene for the coat p r o t e i n is t r a n s l a t e d at a m u c h higher rate than the gene for p r o t e i n A and, apparently, for the replicase gene. C o m p a r i s o n of the amino acid coding tables for these three proteins shows that all codons are used more or less equally in c o d i n g for p r o t e i n A and replicase though this is not so in the case the coat protein. For ex~ample, only two out of six codons are used for e n c o d i n g arginine, one and the same codon UAC codes for all four tyrosines, and the codon AUA is not used at all in coding for eight isoleucines. The authors believe that the p e c u l i a r coding of these three a m i n o acids is due to the necessarily accelerated tran:sl~ation of the gene for the coat protein. It is k n o w n , for example, that the isoaccepting fraction r e c o g n i z i n g the codon AUA in E. colt constitutes only 5 per cent of the total tRNA TM [143]. This m a y c o n s i d e r a b l y i m p a i r syn thesis of p r o t e i n A and replicase for w h i c h the cod.on AUA is used 7 and 12 times, respectively. At the same times, synthesis of the coat protein would not be h a m p e r e d . Min Jou el al. found several m u t a n t s of MS-2 cont a i n i n g the s p o n t a n e o u s silent m u t a t i o n Met --> Ile in the position 108' of the coat protein. Since the t r a n s i t i o n (G --> A) is m u c h more p r o b a b l e in spontaneous m u t a t i o n s t h a n the t r a n s v e r s i o n (G --~ U, C), one might expect the codon AUA for isoleucine (AUG --> AUA) to appear in this place. In the light of the above evidence, such a m u t a t i o n must be lethal. Analysis of nucleotide sequences in the three m u t a n t s revealed the transvers,ion AUG AUU to occur in all the cases. This result confirmed the sin)position of the authors that the codon AUA plays a m o d u l a t o r y role in the bacteriophage MS-2 RNA [144].

Direct experimental evidence confirming participation of tRNA in regulation of protein synthesis. So far we discussed only i n d i r e c t data in favour of tRNA p a r t i c i p a t i o n in the regul.ation of p r o t e i n synthesis. More direct e v i d e n c e comes from exper i m e n t s in w h i c h t R N A p r o l m r t i o n s in a cell or in a cell-free system are deliberately changed by the

t R N A in regulation of biosynthesis.

333

TABLE I.

3"he third

~-glohin

Proinsulin

Growth hormone

Chorionic somatomammotropin

0valbumiu

SV40

~bX-I 7~

MS-2

U

28.7

21.4

16.8

10.1

26.7

40.8

42.8

25.9

C

27.4

33.7

40.6

44.6

25.9

13.2

20 1

30.2

A

6 2

10 2

8.4

10.1

36.4

27.3

15.2

19.5

G

37.7

34.7

34.2

35 2

21

6

18.7

21.9

24.4

position

of codon (pour cent)

NUA

0

1

3

0

12

41

34

67

28

24

20

13

41

51

102

56

2

3

4

6

37

49

47

59

1

3

4

7

1

0

66

72

7

8

8

6

37

76

101

52

19

30

23

15

31

43

99

76

(times) NUG

NCA (times) NCG

NAA (times) NAG

i n v e s t i g a t o r thus m o d i f y i n g the c h a r a c t e r of protein synthesis. F o u r series of such e x p e r i m e n t s can be f o u n d in the literature.

1. Synthesis of cuticular protein in T e n e b r i o molitor. In 1970 Ilan et al. [145] r e p o r t e d the results of a study on th.e d e v e l o p m e n t of Tenebrio molitor. I n t e n s i v e synthesis of the c u t i c u l a r p r o t e i n w i t h a high ,content of t y r o s i n e started in the e p i d e r m a l cells by the 5"7th day of the l a r v a l stage. T y r o s i n e and l e u c i n e l a b e l e d w i t h different isotopes w e r e i n c o r p o r a t e d in vitro into p o l y p e p t i d e s orL polys o m e s isolated at v a r i o u s stages of the larval development. T h e T y r / L e u radi'oactivity ratio in the p o l y p e p t i d e s was 0.25 w h e n polysom,es, tR~N~Aa n d the n e c e s s a r y e n z y m e s w e r e isolated on the first day of t h e l a r v a l stage, a n d 1.64 in p r e p a r a t i o n s isolated on the s e v e n t h day of the d e v e l o p m e n t . T h e authors used v a r i o u s c o m b i n a t i o n s of polysomes fi.e. mRNA), tRNA and enzymes isolated on the 1st and 7 th day of the d e v e l o p m e n t . T h e mRNA n e c e s s a r y for the synthesis of the t y r o s i n e

BIOCHIMIE, 1979, 61, n ° 3.

r i c h p r o t e i n was f o u n d on p o l y s o m e s by the first day, but its t r a n s l a t i o n r e q u i r e d the tRNA and e n z y m e s isolated on the 7th day. Vice versa, the level of t y r o s i n e i n c o r p o r a t i o n in the p o l y s o m e s iso~lated on the 7th day w a s not h i g h w h e n at least one of the t w o r e m a i n i n g c o m p o n e n t s w a s taken on the 1st day. Moreover, the tRNA isolated on the 7th day a n d a m i n o a c y l a t e d to saturation by the enzymes of the first day could i n c o r p o r a t e c o m p a r a b l e amounts of t h e same ,amino acid upon a d d i t i o n of the enzyme p r e p a r a t i o n o b t a i n e d on the 7th day. Apparently, the tRNA of the 7th day c o n t a i n e d isoacc e p t i n g f r a c t i o n s whi,ch could be a m i n o a c y l a t e d only by the e n z y m e s a p p e a r i n g at the same time. These f r a c t i o n s ~> t r a n s l a t i o n of the mRNAs stored in adva.nce for the m a t u r e cuticular protein.

2. Synthesis of ovalbumin in chicken oviduct under the action of estrogen. S h a r m a et al. [146] used a d i r e c t a p p r o a c h : they i n t r o d u c e d e x o g e n o u s tRNA into intact l i v i n g cells.

334

L. A. O s t e r m a n .

E s t r o g e n w a s i n j e c t e d i.m. to c h i c k e.ns for s e v e r a l days. The o v i d u c t was then excised, cut into pieces, a n d i n c u b a t e d for 3-5 h o u r s in a n u t r i e n t m e d i u m c o n t a i n i n g r a d i o a c t i v e amino acids. U n d e r the action of estrogen, the o v i d u c t s t a r t e d to s y n t h e s i z e o v a l b u m i n w h i c h was a s s a y e d by i m m u n o p r e c i p i t a t i o n . In the c o n t r o l e x p e r i m e n t , the b e g i n n i n g of the synthesi~s could be d e t e c t e d four d a y s a f t e r the i n j e c t i o n w h e n o v a l b u m i n c o n t a i n e d 5 p e r cent of the label i n c o r p o r a t e d into soluble p r o t e i n s . This a m o u n t r i s e d to 2,5 p e r cent six d a y s after the injection, a n d to 80-8'5 p e r cent ( t y p i c a l of the hen) after seven days. In the e x p e r i m e n t a l series, tRNA i s o l a t e d f r o m the o v i d u c t of h e n w a s a d d e d at a c o n c e n t r a t i o n of 2:50 ~ g / m l to the n u t r i e n t m e d i u m . The exogenous tRNA p e n e t r a t e d into the c h i c k e n o v i d u c t cells a n d t h e r e u p o n a c c e l e r a t e d ov,albumin s y n t h e s i s at the e a r l y stages of h o r m o n a l stimulation. No~w o v a l b u m i n could b e d e t e c t e d t h r e e days after the i n j e c t i o n of estrogen ( ~ 5 p e r cent). Its level r e a c h e d 3,0 p e r cent four days. after the stimulation, 50 p e r cent after six days, and b e c a m e maximal (as in the control) after seven days. I n t r o d u c tion of tRNA from r o o s t e r liver h a d no effect n e i t h e r h a d the r i b o n u c l e a s e hydrolys.ate of tRNA from the o v i d u c t of hen. Therefore, in the p r e s e n c e of estrogen, the rate of s y n t h e s i s of m R N A for ovalbum'in is h i g h e r t h a n that of the s y n t h e s i s or a c t i v a t i o n of the specifi,c tR'NA f r a c t i o n s necess,ary for t r a n s l a t i o n of this mRNA ; it is t h e c o n t e n t of these tRNAs w h a t limits the rate of o v a l b u m i n synthesis. In t h e i r f u r t h e r stud'ies, S h a r m a et al. [1473 c o m p a r e d , in the same system, the effects of introdtming two e x t r a n e o u s tRN~As (from the liver a n d h e p a t o m a of rat) into the n u t r i e n t m e d i u m . The first li;ke the tRNA from r o o s t e r Iiver, h a d no effect on t h e d y n a m i c s of owalbumin synthesis. In contrust, the tRNA from h e p a t m n a r e p r e s s e d ovalbum i n s y n t h e s i s b y 75 p e r cent, though h a r d l y influenced t h e level of overall p r o t e i n synthesis. The h e p a t o m a tRNA i n d u c e d s y n t h e s i s of o v a l b u m i n w h i c h differed s o m e w h a t in elect r o p h o r e t i c m o b i l i t y and w a s not r e c o g n i z e d by the a n t i s e r u m against n o r m a l ovalbumin. The effect w a s f o u n d to be b r o u g h t about not b y the w h o l e tRN'A f r o m h e p a t o m a but b y its small fraction. T h e results of one m o r e e x p e r i m e n t m a d e by S h a r m a et al. [148] are w o r t h m e n t i o n i n g though, s t r i c t l y speaking, t h e y p r o v e a d a p t a t i o n of a tRNA f a m i l y r a t h e r t h a n regulation. O v a l b u m i n is synthesized i~n the cell-free system of p r o t e i n syn-

BIOCHIMIE, 1979, 61, n o 3.

thesis on the E h r l i c h ascite r i b o s o m e s b y heterologous tR.NAs of the ascite and r a b b i t l i v e r using the mRNA of hen oviduct. Ho~vever, r a b b i t reticulocytes tRNA cannot synthesize o v a l b u m i n in the same system ; instead, it p r o d u c e s f r a g m e n t s w i t h a m o l e c u l a r w e i g h t t w i c e .as low. A p p a r e n t l y , t r a n s l a t i o n of mRNA for o v a l b u m i n stops at a c o d o n 'which is u n c a p a b l e of r e c o g n i z i n g the tRNA of reticulocytes. (That this tRNA is .native is confirmed b y its c a p a c i t y to synthesize globin in the same system using globin mRNA). U n d e r these c o n d i t i o n s y e a s t tRNA c a n n o t at all synthesize p o l y p e p t i d e s . Nevertheless, the m i x t u r e of yeast a n d r e t i c u l o c y t e tRNA can p r o d u c e o v a l b u m i n in the same system. Pre.sum'ably, tl~e tRNA of reticulocytes uses ,a f r a c t i o n of the yeast tRNA w h i c h is n e c e s s a r y for t r a n s l a t i o n of the o v a l b u m i n mRNA.

3. Elimination of the actinomycin blocking of hemoglobin synthesis in chicken embryos. VCainwright a n d Wai,nwright L149-152] have s h o w n that the b l o c k i n g of h e m o g l o b i n s y n t h e s i s b y a c t i n o m y c i n D in i n c u b a t e d b l a s t o d i s c s of chicken e m b r y o s ,can be e l i m i n a t e d b y i n t r o d u c i n g exogenous tRN,As into the n u t r i e n t m e d i u m . The mRNA for globin 'is p r e s e n t at the earliest stages of the e m b r y o n i c d e v e l o p m e n t , but h e m o g l o b i n c a n n o t be s y n t h e s i z e d due to t h e absence of heme. The s y n t h e s i s of heine is t r i g g e r e d once its precursor, 8 - a m i n o l e v u l i n i c acid, a p p e a r s . The mRNA for s y n t h e t a s e of 8-aminolevuli:nic a c i d is p r o d u ced at the e a r l y stage of development, p r i o r to the first somite, and h e r e the f o r m a t i o n of h e m o g l o b i n is b l o c k e d b y a c t i n o m y c i n D at a c o n c e n t r a t i o n of 2 ~ g / m l (this c o n c e n t r a t i o n r e p r e s s e s s y n t h e s i s of mRNA r a t h e r tharL tRNA).. Moreover, ,at the stage of six somites, a,ctinomycin D blocks s y n t h e s i s of 5 - a m i n o l e v u l i n i c a c i d a n d h.emoglobin only beginning w i t h a c o n c e n t r a t i o n of ~0 ~g/ml w h i c h inhibits s y n t h e s i s of tR,NA. T h e r e f o r e , the p r o d u c t i o n of h e m o g l o b i n is b l o c k e d o w i n g to to the i n h i b i tion of s y n t h e s i s of specific tRNAs n e c e s s a r y for t r a n s l a t i n g the mRNA of s y n t h e t a s e of 8-aminol e v u l i n i e acid, since the total content of tRNAs in the e m b r y o n i c cells is h i g h enough. It is at this stage of the e m b r y o n i c d e v e l o p m e n t that the a u t h o r s s t u d i e d in detail the e l i m i n a t i o n of a s t r o n g ~actinomycin b l o c k (100 ~g/ml) by a d d i n g to the m e d i u m exogenous tRNAs isolated f r o m the vitelline m e m b r a n e of .normal five-day-old e m b r y o s o r f r o m chiclcen liver. Selective inactivation w i t h p e r i o d a t e o x i d a t i o n has s h o w n that only a l a n i n e tRNA causes h e m o g l o b i n s y n t h e s i s in the p r e s e n c e of a e t i n o m y c i n D. Moreover, f r a c t i o n a tion on b e n z o y l a t e d cellulose y i e l d e d a m i n o r frac-

t R N A in regulation of biosynthesis. tion (,~ 4 p e r cent) of tRNA Ala w h i c h w a s r e s p o n sible for h e m o g l o b i n synthesis. One is b o u n d to c o n c l u d e t h e r e f o r e t h a t the w h o l e c o m p l e x set of c o m p o n e n t s involved in the p r o d u c t i o n of h e m o g l o b i n (the globin mRNA, the mRNA for s y n t h e t a s e of 8 - a m i n o l e v u l i n i c acid, etc.) is p r e p a r e d in the e m b r y o n i c cells in advance. The u t i l i z a t i o n of these c o m p o n e n t s h o w e v e r is t r i g g e r e d at a c e r t a i n stage of developm e n t by the synthesis of one, and only one, specifi.c (modulator) isoacc.epting f r a c t i o n of tR'NA Aia.

4. Inter[eron story. Although i,nterferon was d i s c o v e r e d over 20 y e a r s ago, the m o l e c u l a r m e c h a n i s m of its action b e c a m e the subject of n u m e r o u s studies only w i t h i n the last t h r e e years.. In most cases, the w o r k is done w i t h cell-free systems of p r o t e i n sy,nthesis in lysates of i n t e r f e r o n t r e a t e d cells in o r d e r to investigate t r a n s l a t i o n of v i r a l RNA in them [153]. S a m u d and J o k l i k [154] have shown that i n h i b i t i o n of v i r a l RNA t r a n s l a t i o n in such systems can be a t t r i b u t e d , in p a r t i c u l a r , to an inhib i t o r of p r o t e i n n a t u r e b o u n d to polysomes. As was r e p o r t e d b y Content et al. [155, 156] and at t h e s a m e time b y Gupta et al. [157], t r a n s l a t i o n of v i r a l mR~NAs is no longer b l o c k e d once a n u m b e r of exogenous tRNAs of a n i m a l l origin are a d d e d to the system. Different f r a c t i o n s of tRNAs are required for such 'an effect in t h e case of di~fferent mRNAs. The m e c h a n i s m of b l o c k i n g the t r a n s l a tion @ith i n t e r f e r o n is v e r y intricate. It invoives t h e p r o d u c t i o n Of a low m o l e c u l a r w e i g h t i n h i b i tor [i58], w h i c h a c t i v a t e s a s p e c i f i c r i b o n u c l e a s e [159], t h e i n h i b i t i o n of i n i t i a t i o n p r o c e e d i n g via p h o s p h o r y l a t i o n of the eIF-2 factor [160, 161], as well as o t h e r p h e n o m e n a m a n y of W h i c h are stim u l a t e d by the p r e s e n c e of the d o u b l e - s t r a n d e d r e p l i c a t i v e form of Viral RNA. H o w e v e r , w e Shall focus our attention on the di~hiockifig effect caused b y t h e a d d i t i o n of tRNA. F a l c o f f el al. [162] stud i e d this e f f e c t for polyl~uCine s y n t h e s i s using syntheti,c oligonucle0tideS as templates. The synthesis is e f f e c t i v e l y i n h i b i t e d - i n tlie lysates of L c e l l s t r e a t e d w i t l i interferon.: The i n h i b i t i o n is -eliinin,ated b y - a d d i n g t h e ~ a j o r fracii,on of y e a s t tRNA ~,, to t h e system. I.n t h e i r f o i l o w i n g ~,onk [i63], t h e s e - a u t h o r s t h o r o u g h l y p u r i f i e d the fraction Of tRNA Le/~ With t h e ainticodon UAG. T h e c o d o n s for this f r a c t i o n a r e ~the t r i p l e t s (~UA and GUG. It w a s f o u n d that tRNA ~ h e l i m i n a t e d the b l o c k i n g of p o l y i e u c i n e s y n t h e s i s o n the t e m p l a t e poly(UC). Therefore, it r e c o g n i z e d the codons CUU and CU,C ; in o t h e r Words, the code for leucine in this case is two-lettered. In view of w h a t has been

BIOCHIMIE, 1979, 61, n ° 3.

335

d i s c u s s e d above, this conclusi(m is not u n e x p e c t e d . It is m o r e diffi,cult to account for the e l i m i n a t i o n of the i n t e r f e r o n b l o c k of t r a n s l a t i n g the templates poly(UG), poly(UA) and even poly(U) .using the same tR'NJAl~" w i t h the a n t i c o d o n UAG r e p o r ted in that w o r k . It is p o s s i b l e that w o b b l e - p a i r i n g at the ,fi.rst c o d o n p o s i t i o n takes place d u r i n g t r a n s l a t i o n of s y n t h e t i c templa.tes. A n o t h e r interp r e t a t i o n is possible, n a m e l y , that tRNALeu posses, "-'" "-UAG ses, a p a r t from the a n t i c o d o n , some other structure p e c u l i a r i t i e s w h i c h m,gk.e it p o s s i b l e to eliminate the i n t e r f e r o n b l o c k i n g of t r a n s l a t i o n in any ease, for any c o d o n s even if t h e y are not e n t i r e l y c o m p l e m e n t a r y . This w i l l be d i s c u s s e d later. In 1976 Z i l b e r s t e i n et al. [164] r e p o r t e d that the b l o c k i n g of t r a n s l a t i o n Of mRNA for M.engo virus and globin w a s e l i m i n a t e d in the lysates of L cells t r e a t e d w i t h i n t e r f e r o n b y the t h r e e t h o r o u ghly purified nfinor i s o a c c e p t i n g fra,ctions of tRNA L~,, from beef Iiver. T h e c o d i n g specificities of these f r a c t i o n s w e r e d e t e r m i n e d from b i n d i n g w i t h ribosOmes in the p r e s e n c e of triplets. F r a c t i o n s A and B p r e d o m i n a n t l y r e c o g n i z e d the codon CU 6, w h e r e a s f r a c t i o n C, c o d o n s of the UU~ type. The f o l l o w i n g facts w e r e also established. ( a ) D e s p i t e the i d e n t i c a l c o d i n g specificitY, f r a c t i o n B w a s far m o r e effective than fraction A in e l i m i n a t i n g the b l o c k i n g Of the M~engo virus RNA t r a n s l a t i o n . (b) In contrast, f r a c t i o n A was m o r e effective than f r a c t i o n B in d e b l o c k i n g the g i o b i n mRNA t r a n s l a t i o n in the same system. (c) F r a c t i o n C w a s less effective i,n both Cases, so that only s h o r t e r p o l y p e p t i d e s w e r e s y n t h e s i T TA zed. A p p a r e n t l y , c o d o n s of the UU G type blocked by i n t e r f e r o n are l o c a t e d closer to the p o i n t w h e r e t r a n s l a t i o n begins. (The h i g h effectiveness of f r a c t i o n s A and B in e l i m i n a t i n g the interferon block m e a n s that t h e y can, also d e b l o c k p r o x i m a l c o d o n s of the UU~ type, t h o u g h these f r a c t i o n s are most specific t o w a r d the c o d o n CUG. This situation is s i m i l a r to that w h i c h w,as discussed for the p r e v i o u s w o r k ) . (d) The m a j o r fraction of tRNA r~" from w h i c h f r a c t i o n A w a s removed could not e l i m i n a t e the i n t e r f e r o n b l o c k i n g of the globin mRNA t r a n s l a t i o n t h o u g h it recognized the same c o d o n CUG as f r a c t i o n A. This last observation., as w.ell as c o m p a r i s o n of the t w o f o r m e r Ones, suggest t h a t the c a p a c i t y to e l i m i n a t e the i n t e r f e r o n blo,ck in t r a n s l a t i o n is caused b y s o m e t h i n g m o r e than a c e r t a i n c o d i n g specificity of tRNA. F r a c t i o n s A, B a n d C, a p p a rently, s h o u l d have some a d d i t i o n a l c h a r a c t e r i s tics e n d o w i n g them w i t h a c a p a c i t y to d i s c r i m i nate different mRN~s. It is not u n l i k e l y that these charaCteristics are of a m o r e general significance

336

L. A. O s t e r m a n .

in terms of the regulatory f u n c t i o n of tRNA. This will be discussed belo~v. It w o u l d be necessary to quote the r e c e n t w o r k of Mayr et el. [165] i n w h i c h the RNA of eneephalomyoc~/rditis v i r u s .(EMC,-RNA) i n the lysate of mouse p r o e r y t h r o b l a s t s treated w i t h i n t e r f e r o n could be t r a n s l a t e d only u p o n d e b l o c k i n g caused b y a d d i n g t h e tot,el tRNA of r a b b i t liver. T r a n s lation r e a c h e d 50 per cent of the m a x i m a l level w h e n one of its isoaccepting t R N A ~ u w a s used. This capacity was not lost even u p o n its almost complete oxidation by sodium periodate. Surprisingly it t u r n e d out that tRNA must not necessarily he involved in p o l y p e p t i d e synthesis i n order to remove the interf,eron block. This effect can. be accounted for u s i n g the e x p e r i m e n t a l evidence of t h e authors. W h e n the block i n transeation was p a r t l y e l i m i n a t e d b y m e a n s of native or oxidized tRN~A~ys, n a s c e n t p o l y p e p t i d e s ~vere m u c h shorter t h a n w h e n the total n o n - o x i d i z e d tRNA was added. However, Zilberstein et el. [164] obtained n o r m a l p r o t e i n s w h e n the RN.A of Mengo virus w,as translated, as well as n o r m a l globin, by d e b l o c k i n g u p o n a d d i t i o n of the m i n o r tRN,AT~u fractions. It is possible that the i n t e r f e r o n block in t r a n s l a t i o n of EI~C-RNA i n the e x p e r i m e n t s of Mayr el at. was due to ,ribosomes b e i n g attached to the blocked codon for lysine. Possibly, the native a n d oxidized tRN'ALye molecules only liberate ribosomes t h e r e b y p e r m i t t i n g repeated transl a t i o n at the b e g i n n i n g of the E ~ C - R N A molecule. Yet, to p e r f o r m this function, tRNA~y~ m u s t possess specific properties apart from its capacity to b e aminoacylated. T h e specific characteristics of tR~NAs related to t h e i r r e g u l a t o r y f u n c t i o n s are the subject of the following section.

Development Of the Ames and Hartman modulation hypothesis. Prese!ectiou hypothesis. Not all of the above results can be i n t e r p r e t e d in terms of the m o d u l a t i o n h y p o t h e s i s based entirely on the cod'on-anticodon recognition. Analysis of these results mak.es it possible to put f o r w a r d .certain suggestions Within the scope of the modulation hypothesis ; these will be r e f e r r e d to as the tl~NA preselection hypothesis. However, before f o r m u l a t i n g the m a i n concepts o f Ibis hypothesis let us discuss two series of e ~ p e r i m e n t a l evidence. First, w e shall dwell on -the m u l t i p l i c i t y of isoaccepting tRNAs and the n a t u r e of their diffevence. J.uarez el el. [166~, by c o m b i n i n g the t e c h n i q u e s of tRN~A chromatographic separation on I~D-cellulose and i n reversed p h a s e s , obtained seven isoaccepting fractions of

BIOCHIMIE, 1979, 61, n ° 3.

tRNALys from mouse fibroblasts. Only two codons c o r r e s p o n d to lysine. DeLeon et el. [167] c o m p a r e d the c h r o m a t o g r a p h i c profi,les of tRNA m~ (also two codons) for six different organs of mouse. Six peaks in the same position (whose p r o p o r t i o n s val'ied a c c o r d i n g to the organs) were registered in all the cases. Olsen and P e n h o e t [168] compared the isoaccepting profiles of tRNA T-vr f,rom placenta ,and c a r c i n o g e n i c cells (HeLa). In both cases, there were four isoaccepting fractions for each of the two t y r o s i n e codons. T h e i r p r o p o r t i o n s however were different and the profile for tRNATr r from the HeLa cells was displaced d u r i n g chro.matography in reversed phases as c o m p a r e d to the profile for placenta. I n contrast, the profiles for p l a c e n t a and n(~rmal liver h a d identical position though they v a r i e d in the p r o p o r t i o n s of peaks. It is n o t e w o r t h y that all three lines of evidence were o b t a i n e d w i t h a m i n o acids whose codons are in the t h i r d c o l u m n of the code table. This gives ground, as has been m e n t i o n e d before, to c o n s i d e r the respective IRNAs as p r o b a b l e candidates for p e r f o r m i n g the regulatory functions. F i n a l l y , Richer [1~9] r e c e n t l y separated three isoaccepting tRNA Met from r a b b i t reticulocytes. One of them t u r n e d out to be an i n i t i a t o r but the r e m a i n i n g two i n c o r p o r a t e d m e t h i o n i n e into g.olb i n p o l y p e p t i d e chains. The effectiv.eness of their utilization in the homological cell-free system of globin synthesis differed more t h a n four fold. (N.ote that some other p r o t e i n s are synthesized in reticulocytes apart from globin). The above d,ala i n d i c a t e that the difference b e t w e e n i s o a c c e p t i n g fractions is not an artifact of tR'NA isolatvion, 'a'nd c a n n o t be attributed only to the diversity in a n t i c o d o n s ; instead, it stems from other s t r u c t u r a l differences b e t w e e n the molecules. The v a r i e t y of these differences was m e n t i o n e d above. SO~etimes the l o c a t i o n of m i n o r bases is u n u s u a l , e.g. m2G in the a c c e p t o r stem [170] or X in the D loop [83]. The supressor mutation of tRNAT'y was f o u n d i n E. colt [171]. As a result of the substitution G --* A in the stem of the D loop, the t r y p t o p h a n a n t i c o d o n O2A a c q u i r e d the capacity to recognize the t e r m i n a t i o n codon UGA in this case. As was found recently, the s,ame supressar tRNA"try can recoznize the codon UGU as well [172]. These two findings c o n t r a d i c t the w o b b l e hypothesis, but fit in w i t h the two-letter code. What is i m p o r t a n t is that this u n u s u a l recog n i t i o n of codons is caus,ed by s u b s t i t u t i o n of only one base in a tRN~A molecule far off from the anticodon, thus b e i n g the result of a s t r u c t u r a l rearrangement. Let us now c o n s i d e r certain i n f o r m a t i o n on n o n ribosomal p r o t e i n s involved in the t r a n s l a t i o n

t R N A in regulation of biosynthesis. apparatus. It is well k n o w n f a c t that w a s h i n g w i t h 0.5 M KC1 removes from the polysomes of eukaryotes a l a r g e n u m b e r of p r o t e i n s h a v i n g various size a n d electrophoretic mobility. These in,clude p~rotein t r a n s l a t i o n factors. Data are avai= lable to the effect that aminoacyl-tRNA syntherases and, possibly, IRNA m o d i f y i n g enzymes are c o n n e c t e d w i t h ribosomes. However, the s p e c t r u m of physiological f u n c t i o n s of these various, p r o t e i n s is still broader. As has been m e n t i o n e d above, the p r o t e i n i n h i b i t o r of t r a n s l a t i o n p r o d u c e d u n d e r the action of i n t e r f e r o n is b o u n d to polysomes a n d c a n be w a s h e d w i t h 0J5 M! KC1. Rosenfeld a n d B~rrieux [173] f r a c t i o n a t e d p r o t e i n s o b t a i n e d u p o n w a s h i n g the polysomes w i t h 0.,5 M KC1 by c h r o m a t o g r a p h y on phosphocellulose. Many of these c o u l d be selectively attached to mRNA (but not to tRNA or r i b o s o m a l RNA). It it likely that part of these p r o t e i n s is w e a k l y b o u n d to mRNA in vivo. Moreover, a n u m b e r of p r o t e i n s firmly attached to mRNA can be f o u n d in both i nformosomes and mRNP f u n c t i o n i n g in the polysomes. Two of them ,('M,W ~2,000 and 78,000) seem to be the same i n different cells of a n i m a l origin [174]. Apparently, they m'e necessary for d i s p l a y i n g the template activity of mRNA,. Apart from these, several other proteins fiirmly b o u n d to mRNA are detected b y electrophoresis in p o l y a c r y l a m i d e gel [175]. T h i r t y p r o t e i n l i n k e d to the mRNA of reticulocytes have been separated u s i n g t w o - d i m e n sional electrophoresis. Half of these c a n n o t be r e m o v e d even w i t h 1 M KC1. The p o p u l a t i o n of p r o t e i n s changes among different a n i m a l s !1751. As was s h o w n by L i a u t a r d a n d KShler [1771, the electrophoreti'c mobilities of mRNPs separated from HeLa ribosomes u n d e r very mild c o n d i t i o n s are i d e n t i c a l in a sucrose g r a d i e n t despite the fact that their- d i m e n s i o n s are highly h,eterogeneous. The authors believe that the p o p u l a t i o n of proteins is repeated along the length of the mRNA molecule. R,e.cently S p i r i n [1781 has put f o r w a r d a c o n c e p t a c c o r d i n g to w h i c h the m a j o r i l y of proteins involved in ,translation are firmly b o u n d to mRNA in i n f o r m o s o m e s and actively t r a n s l a t e d mRN,I~ in polysomes. He refers not only to the t r a n s l a t i o n factors a n d m o d i f y i n g enzymes, but also to the factors r e g u l a t i n g the rate of t r a n s l a t i o n . This concept plays an i m p o r t a n t role in the preselection hypothesis discussed below. K u r l a n d et al. [179] s~lggested an allosteric mec h a n i s m for the codon d e p e n d e n t selection of tRN~A in ribosomes. A c c o r d i n g .to this hypothesis, the correct c o d o n - a n t i c o d o n i n t e r a c t i o n serves as an allosteric effector d e t e r m i n i n g i n the total con-

BIOCHIMIE, 1979, 61, n ° 3.

337

f o r m a t i o n of a tRNA molecule changes w h i c h o p e n its non-specific sites of b i n d i n g w i t h ribosomes. This modified c o n f o r m a t i o n is then stabilized by the i n t e r a c t i o n of tRNA with a ribosome a n d mRN'A. Though events discussed below occur p r i o r to the c o d o n - a n t i c o d o n i n t e r a c t i o n , the possibility of d y n a m i c slructural-confo:rmational transformalions of the translat'ion m e c h a n i s m will be of major i m p o r t a n c e . Let us first formulate the basic suppositions.

1. Possible distribution of all tRNAs among the finite number of conformational groups. The spatial c o n f o r m a t i o n of each tRNA molecule w o u l d be d e t e r m i n e d by its p r i m a r y structure. The v a r i a b i l i t y of p r i m a r y structures d e t e r m i n e s a great m u l t i p l i c i t y of conformations. However, the u n i v e r s a l c h a r a c t e r of the clover-leaf model for the s e c o n d a r y tRN~A structure suggests a::cer tai'n s i m i l a r i t y 'in spatial configurations. The n a t u r e a n d p o s i t i o n of modified nucleosides make a c o n s i d e r a b l e c o n t r i b u t i o n to both c o n f o r m a t i o n of a tRNA molecule and, p a r t i c u l a r l y , to the physical p r o p e r t i e s of its conta.ct surface. As has been noted above, c e r t a i n m i n o r bases occupy fixed positions, in p a r t i c u l a r , in the c e n t r a l part of a tRNA molecule w h i c h seems to play the key role in p r o d u c i n g its outer contacts. Consequently, there might be a finite n u m b e r of v a r i a n t s in the d i s t r i b u t i o n of methyl groups w i t h i n this region.

Presumably, all tRNA molecules can be subdivided into a finite n u m b e r of groups a c c o r d i n g to the general c h a r a c t e r of spatial configuration and the d i s t r i b u t i o n of methyl groups on contact surfaces. The n u m b e r of groups and their composition are d.etermined by the value of allowed spatial differences b e t w e e n molecules similar in conf o r m a t i o n w i t h i n each group. If these criteria are applied more strictly, the n u m b e r of tRNA molecules in a group diminishes. The d i s t r i b u t i o n a m o n g the c o n f o r m a t i o n a l groups does not d e p e n d directly on the a m i n o acid a n d coding specificity of tRNA. F o r instance, even those isoaccepting tRNAs w h i c h recognize one and the same codon can belong to different groups. 2. Possible existence of a finite number of definite spatial ribosomal conformations. There is some g r o u n d to believe, p a r t i c u l a r l y in order to i n t e r p r e t the above e x p e r i m e n t a l data, that ribosomes can also a c q u i r e a finite n u m b e r of different and definite c o n f o r m a t i o n s due to

L. A. O s l e r m a n .

338

changes i n the mutual a r r a n g e m e n t of t h e i r proteins a n d RN'A as weI1 as p r o t e i n s b o u n d to ribosomes. The f u n c t i o n a l role of such t r a n s f o r m a t i o n s in the structure of ribosomes w o u l d he to p r o v i d e favourahle c o n d i t i o n s for tRNA molecules, belonging to a given c o n f o r m a t i 0 n a l group, to penetrate into a ribosome, be p r o p e r l y oriented in it, and realize c o n t a c t of the a n t i c o d o n with the codon of mRNA. Therefore, we postulate a possibility of tRNA molecules .to be preselected by ribosomes accordi:ng to their c o n f o r m a t i o n a l groups. The presele.ction can be more o r less strict, thus d e t e r m i n i n g the composition of a selected tR~NA group.

3. Factors controlling structural rearrangements o[ ribosomes. These factors m a y i n c l u d e : (a) mRN~A codons or even a base of each new codon e n t e r i n g a ribosome in the course of translation ; (b) proteins b o u n d to m R N A ; (c) p r o t e i n s attached to ribosomes, i n c l u d i n g regulatory p r o t e i n s from the cytoplasm.

4. The role o[ codon-anticodon recognition. I n t e r p r e t a t i o n of the role of c o d o n - a n t i c o d o n r e c o g n i t i o n for selection a m o n g iRNAs b e l o n g i n g to the same preselected group is the same. This is also true of all the s u b s e q u e n t events of translation, and the above m e n t i o n e d hypotheses of F e l d m a n and K u r l a n d can be applied. Thus, the m o d u l a t i o n hypothesis of tRNA participation i n regulati:ng p r o t e i n synthesis should be s u p p l e m e n t e d w i t h the hypothesis of tRNA preselection a c c o r d i n g to its spatial c o n f o r m a t i o n . However, p r i o r to discussing the m e a n i n g of this supplement, it w o u l d be relevant to note that the preselection hypothesis also accounts for the high rate of p r o t e i n synthesis. Indeed, if a m i n o acyl-tRNAs were selected only by codon recognition, dozens of t h e i r molecules w i t h i m p r o p e r a n t i c o d o n s should be tested a n d rejected for each step of an a m i n o acid b e i n g attached to a polypeptide. However, each testing implies not only p e n e t r a t i o n b u t also definite o r i e n t a t i o n of tRNA in a ribosome. All this t~kes time. It is obvious that the two-step selection a c c o r d i n g to the scheme b e i n g p r o p o s e d w o u l d be far more e c o n o m i c a l and r a p i d (just l~k.e Ifmding a book u s i n g a c a t a -

BIOCHIMIE,

1 9 7 9 , 61, n ° 3.

logue is faster t h a n looking through al the books in a library). The m u l t i p l i c i t y of tRNA isoaccepting fractions can be i n t e r p r e t e d in terms of the preselection hypothesis. A p p a r e n t l y , some of the fractions h a v i n g i d e n t i c a l codons belong to different conform a t i o n a l groups, a n d this may be ,of key importan.ce i n .regulating p r o t e i n synthesis. W i t h i n the scope of the Ames ~ H a r t m a n hypothesis, the m o d u l a t o r y tR'NA .(limiting the rate of p r o t e i n synthesis) might differ from other isoaccepting tRNAs only i n its a n t i c o d o n . Now we may assume that this difference can be also caused, in a n u m b e r of cases, b y its c o n f o r m a t i o n , i.e. the tRNA belongs to the c o n f o r m a t i o n a l group w h i c h is the o m y one to be used for t r a n s l a t i n g a given codon in a given mRNA. Such an i n t e r p r e t a t i o n implies that selection of tR,NAs a n d regulation of p r o t e i n synthesis also involve mRNA molecules via specific proteins b o u n d to them. The p r o t e i n s of mRNP m a y control sele.ction of the tRNA c o n f o r m a t i o n a l group, as well as the strictness of this selection, by .acting on the c o n f o r m a t i o n of a ribosome or particip a l i n g w i t h it in the f o r m a t i o n a definite spatial complex. A similar, though i n d e p e n d e n t function, can be fulfilled by the regulatory p r o t e i n s of the cytoplasm w h i c h are b e i n g attached to ribosomes, as in the case of the i n t e r f e r o n i n h i b i t o r in translation of viral ' ~ A s . All in all, this is a flexible m u l t i c o m p o n e n t system capable of regulating the ratio b e t w e e n the rates of t r a n s l a t i o n of different mRNAs. The above c o n s i d e r a t i o n s do not reduce the significance of the m e c h a n i s m of c o d o n - a n t i c o d o n r e c o g n i t i o n i n c l u d i n g the t h i r d codon base. Its role ( w i t h i n the frame of the conform a t i o n a l group) is s u b s t a n t i a t e d by the previously discussed characteristics Of the a m i n o acid coding tables, or by the correlation between the growth rates for a n u m b e r of hepatomes and a decrease in the selectivity of coding at the expenses of i n h i b i t i n g the t h i o l a t i o n of their tRNA at the first a n t i c o d o n base [1251. However, the completeness of .an a n t i c o d o n being complem e n t a r y (three-letter recognition) has advantages only of a Mnetic, competitive character. A codon .can be recognized also by two letters if compet i t i o n is excluded .as a result of the c o n f o r m a t i o n a l p r e s e l e c t i o n or i n a system in vitro c o n t a i n i n g only one isoaccepting tRNA fraction [121, 163]. Yet, the c o n f o r m a t i o n a l preselection should be also of a competitive rather t h a n absolute c h a r a c t e r as follows from the data on the viability of submethylated E. colt strains and their r e p l a c e m e n t w i t h a wild strain u p o n c o m b i n e d c u l t i v a t i o n [93].

tRNA

in regulation

F u r t h e r s u p p o r t c o m e s f r o m th.e f a c t t h a t i n c o r p o r a t i o n of a m i n o a c y l - t R N A of a n i m a l o r i g i n i n t o p o l y s o m e s is d e c e l e r a t e d r a t h e r t h a n c o m p l e t e l y b l o c k e d on i n h i b i t i n g the m e t h y l a t i o n of t R N A [108]. S p e c i a l i z a t i o n of t h e t R N A Ala in which o n l y o ~ e , n u c l e o t i d e w a s substiCuted in t h e e x p e r i m e n t s of Meza et al. [7] c a n be easily e x p l a i n e d w i t h i n t h e s c o p e of the a b o v e hypothesis. The hypothesis accounts for the r o l e of a s p e c i f i c tRNALy s f r a c t i o n t y p i c a l of r a p i d l y p r o l i f e r a t i n g tissues in t h e e x p e r i m e n t s of O r t w o r t h et al. [19]. It is l i k e l y t h a t t h e s e t R N A s d i f f e r in t h e c o n f o r m a t i o n a n d p o s i t i o n of m i n o r nucleotides from other isoaccepting tRNAs with identical anticodons, and can therefore perform a p a r t i c u l a r r e g u l a t o r y f u n c t i o n . T h e s a m e is t r u e of t h e r e s u l t s o b t a i n e d b y H i r s h [171] w h o s t u d i e d t h e s u p p r e s s i o n of a n o n s e n c e m u t a t i o n u s i n g tRNATry w i t h t h e a n t i c o d o n CCA. A p p a r e n t l y , t h e o n l y s u b s t i t u t i o n G ~ A in t h e D l o o p of t h e t R N A m o l e c u l e c a u s e d s u c h a p r o f o u n d c h a n g e in its c o n f o r m a t i o n t h a t t h e t e r m i n a t i o n c o d o n UGA c o u l d be r e a d as .a t r y p t o p h a n c o d o n by t h e first t w o letters. T h e h y p o t h e s i s is c o m p a t i b l e w i t h t h e r e s u l t o b t a i n e d b y Z i l b e r s t e i n el al. [164] w h e r e t h e i n t e r f e r o n b l o c k c o u l d be e l i m i n a t e d by o n l y one m i n o r f r a c t i o n o f t R N A I ~ w h i c h s e e m e d to possess t h e r e q u i r e d s p a t i a l c o n f i g u r a t i o n . H e r e , the m a j o r t R N A L~" t h o u g h h a v i n g t h e s a m e c o d i n g s p e c i f i c i t y falls w i t h i n t h e t R N A c o n f o r m a t i o n a l g r o u p u n d e r the .action of i n t e r f e r o n (for v i r a l o r g l o b i n ml~NA). T h i s t R N A g r o u p r e m a i n e d (< a l l o w e d >> for its o w n m R N A due to a d i f f e r e n c e in t h e c o m p o s i t i o n of m R N P p r o t e i n s . It is also l i k e l y t h a t t h e a c c e l e r a t e d g r o w t h a n d d i f f e r e n t i a t i o n of m a l i g n a n t d e n o v o f o r m a t i o n s are c a u s e d b y a less s t r i c t c o n f o r m a t i o n a l s e l e c tion.

Conclusion. T h e a b o v e a n a l y s i s of e x p e r i m e n t a l e v i d e n c e suggests t h a t t h e c o n c e p t a b o u t t h e p a r t i c i p a t i o n of t R N A s in the r e g u l a t i o n of p r o t e i n b i o s y n t h e s i s at t h e t r a n s l a t i o n a l l e v e l is still a w o r k i n g h y p o thesis. D i r e c t d a t a s u p p o r t i n g o f it a r e still insuff i c i e n t a n d c a n b e s~bj,ected to c r i t i c i s m . But t h e c o n c e p t of t R N A c o n f o r m a t i o n a l p r e s e l e c t i o n ~ c c o u n t s for t h e r e s u l t s of c e r t a i n e x p e r i m e n t s w h i c h c a n n o t be i n t e r p r e t e d in t e r m s of the Ames a Hartman modulation hypothesis. However, BIOCHIMIE, 1979, 61, n ° 3.

339

of biosynthesis.

f u r t h e r e x p e r i m e n t s a r e n c e s s a r y to c o n f i r m t h i s concept. N e v e r t h e l e s s , a p o s s i b i l i t y of t R N A b e i n g i n v o l v e d in the r e g u l a t i o n of p r o t e i n s y n t h e s i s is of major importance and practical significance, thus c a l l i n g for f u r t h e r s t u d i e s in this field of r e s e a r c h .

LITERATURE. 1. Ames, B. N. & I-Iartman, P. (19~3) Cold Spr. Harb. S y m p . Quant. Biol., 28, 569-578. 2. Sueoka, N. ~ Kano-Sueoka, T. (1964) Proc. Nat. Acad. Set., 52, 15~5-1540. 3. L~t~auer, U. Z. ~ Inouye, H. (107~) in A n n u a l Bey. Bioch., (Snell, E. E., ed.) vol. 42, pp. 439470. 4. Garel, J. P., Mandel, P., Chavancy, G. & Daillie, J. (1970) FEBS Letters, 7, 327'-329. 5. Chavancy, G., Garel, J. P. & Daillie, J. (1975) FEBS Letters, 49, 38D-384. 6. Araya, A., Kr~us,kopf, M. a Siddiqui, M. A. Q. (1975) Biochem. Biophys. Res. Commun., 67, 924-934'. 7. Meza, L., A~aya, A., Leon, G., Krauskopf, M., S4ddiqui, M. A. Q. & Garel, J. P. (1977) FEBS Letters, 77, 255-2~0. 8. Hentzen, D., Garel, J. P. a Keith, G. (1976) Biochem. Biophys. Res. Commun., 71, 241-248. 9. Suzuki, Y. ,~ Brown, D,. D. (1972) J. Mol. Biol., 63, 409-42'9 10. Fourn~ier, A., Gh~avancy, G. & Garel, J. P. (1976) Biochem Biophys. Res. Commun., 72, 1187-1194. ll. Beck, G., H er~tzen, D. & El)el, J. P. (1970) Biochim. Biophys. Acta, 213, 55-67. 12. Klyde, B. J. & Bernfield, M. R. (1973) Biochemistry, 12, 3757-3763. 13. Gerlinger, P., Le Meur, M..-A. & Ebe,1, J.-P. (1975) FEBS Letters, 49, 376-379. 14. Le Mleur, M.-A, Gerlinger, P. a Ehel, J.-P. (1976) Eur. J. Bioehem., 67, 519-526. 15. lllinger, D., Le Me ur, M.-A., Geding.er, P. & Ebe'l, JL-P. (1974) Biochimie, 56, 529-53L 16. Sharma, O. K., Beezley, D. N. & Borek, E. (1976) Nature, 262, 62-63. 17. Vi.Otti, A., B~tduoci, C. a Wei~l, J. ~. (1978) Biochim. Biophys. Aeta, 517, 125-132. 18. Ortworth, B'. J: a Liu, L: P. (1973) Biochemistry, 12, 3978-3984. 19. Ortwor~h, B. J., Yonuschot, G. R. a Garlson, J. V. (1973) Biochemistry, 12, 3985-3991. 20. Juarez, H., Juarez, D., Hedgcoth, C. & Ortworth, B,. J~ (1975)Nature, 254, 359-360. 21. Tidweil, T., Bruce, B. J. a Griffin, A. C. (1972) Cancer Res., 32, 104)2-1098. 22. S~aw, P. A., Tidwell, d. T., Cl~ia, L . L . S . Y . , Ra~nderath, E. a Randera.t~. K. (1978) Biochim. Biophys. Acta, 518, 459-463. 23. Thom,ale, J. & Nass, G. (1978) Eur. J. Biochem., 85, 407~-418. 24. Rogg, H., Miiller, P., Keith, G. & Staehelin, M. (1977) Proc. Nat. Acad. Sci., 74, 4243-4247. 25. H.atfieid, D. & Port~ugal, F. H. (1970) Proc. Nat. Acad. Set., 67, 1200-1206. 26. Gai~o, R~. C. & P,estka, S~. (1970) J. Mol. Biol., 52, 195-219. 27. Votkers, S,. A. S. ~ Taylor, M. W. (1971) Biochem i s t r y , 10, 44~-497. 28. Gomano, F., CA~iarugi, V. P., Pirro, G. ~ Marini, M. (1971) Biochemistry, 1O, 9/)0-9'08. "23

340

L. A. Osterrnan.

29. Gla~rkvon, S. G. ~ P~unner, M. N~ (1971) Biochim. Biophys. Acta, 238, 498-502. 30. Sriniv,asan, D., Sa'inlvasan, P. R., Grunberger, D., VC3e~inst~in, J=. B~. ~ l~orris, H. P. (19'7'1) Biochemistry, 10, 196.6-197~. 31. B,efort, J. J., Merci.e% ~., P~efort, N., Beck, (L Ebel, J.-P. (19.72) Bioehimie, 54, 1327-1333. 32. Hayashi, 55., Griffin, A. C., Duff, 1~. & Rapp. F. (1973~. Cancer Res., 33, 902-905,. 33. CvalTag~h,er, R. E~, Ting, R. C. ~ Gall,o, R. C. (19'72) Biochim. Biophys. Acta, 272, 568-582. 34. Briseoe, W. T., Syr.eWiez, J. J., l~arsh,al], M. V. Griffin, A. C. (1975) Biochim. Biophys. Aeta, 383, 441-~45. 35. Briseoie, W. T., Taylor, W , Griffin, A, G., Duff, R. Rapp, F. (1972 ~) Cancer Res., 3'2, 1753-1755. 36. Grun,berger, D., Weiinstein, J. P~. ,~ Mumsh~in,ski, J. F. (1975) Nature, 253, 66-67. 37. McGloskey, J. A. ~, Nishimura, S. (1977) Accounts of Chem. Bes., 10, 40,3~410. 38. K~uchino, Y. ~ B~)rek, E. (1976~ Cancer Res., 36, 2~932;-2936. 39. Sharma, O. K. & K~chino, Y. (1977) Biochem. Biophys. Res. Commun., 78, 591-595 40. Mul~erjee, ~. ~ Gol&feder, A. (19'76) Cancer Res., 36, 333~-3338. 41. Merei~er, J., B~for~, N., Beck, O., Ebe], J.-P. ,~ Blefort, J. J. (1972y Biochimie, 54, 1319-1326. 42. Lerm, an, iV[. J , Pilipenko, N. 1%., Ug~rova, T. Y., Sokolova, E. S., Vinnizky, L . J. a Phishkov,a, Z. P. (197'6) Cancer Res., 36, 2995-3900'. 43. Boxek, E., Daliga, tk S., Geh~ke, (2. W., Kuo, C. W., B~elman. S., Troll, W. & W,a[k~es, T. P. (1977) Cancer Res., 37, 3362-3366. 44. F u j i m u r a , S. a SIl~im.izu, M. (1977) Biochem. Biophys. Bes. Commun., 79, 763-768. 25. J,~eobson, E. L., Juarez, IT., II~dgcoth, G. & Gonsigli, R. A. (1974) Arch. Bioehem. Biophys., 163, 666-670. 46. H en~tzen, D. (1976) Cancer Res., 36, ~082-3085. 47. Gate1, J. P., Hentzen, D. ~ Dailli,e, J. (1974~) FEBS Letters, 39, 359-3'63. 48. Smith, R. & LaForrest, H. S. (1973) Developm. Biol., 33, 123-129. 49. Nwagwu, M. ~ Lian~a, J. (1974) Can. J. of Biochem., 52, 838-844. 50. Agris, P. F. (1975)Arch. Biochem. B i o p h y s , 170, 114-123. 51. Pal, atnik, C. IV~., I~t~, E. R. & l~renn~r, M. (1977) J. Biol. Chem., 252'. 69,4-703. 52. Ostermann, L, A~, Sverdlova, P. S. ~ Tshupeeva, V. V. (1977) Ontogenes, 8, ~2&237. (russi,an). 53. Lane~ C. D,, Gurdon, J. B. ~ Woodland, H. R. (1974) Nature, 251, 436-4'37'. 54. White, B. N., Terrer, G~ M~., Holt~en, J. & Suzuki, D. T. (1973:) J. Mol. Biol.. 74, 635-651. 55. Carpousis, A., Ghristner, p . ,~ Rosenbloom, J. (197,7) J. Biol. Chem., 252, 2447-2449. 56. Carpousis, A., Christner, P. ~ Rosenbloom, J. (1977) J. Biol. Chem., 252, 8'02~-8020. 57. Tr~vnicek, ~ . (1969y Biochirn. Biophys. Acta, 182, 42q-439. 58. Wang, S.., Kot~ari, 1~. M., Ta.yl(~r, M. & Hung, P. (1973) Nature, N. B., 242, 133-135. 59. Randerath, K., Rosen~hal, L. J. ~ Zame,cn.ik, P. C. (1971) Proc. Nat. Acad. Sci., 68, 3233-3237. 60. W~ate:rs, L. O. ~ Mu~]l'im I~. G. (1977) in Progr. Nucleic. Acid. Res. & Mol. Biol., (Cohn, W. E. Ed.) v ol. 20, p. 13d. 61. Waters, 'L. G. (1978) Biochem. Biophys. Res. Commun., 81, 822~-8217. 62. Travnicek, M. • Riman, J. (1973) Nature N. B., 241, 6.0-62;. 63. Eldor, K. T. & Sin,itch, A. I~. (1'973) Proc. Nat. Acad. Sci., 70, 282~-2826'. 64. Wang, S., Kothari, R. M., T,aylor, M. W. ~ Hung, P. P. (197'4) Biochim. Biophys. Acta, 340, 52-63. 65. Cl~en. E. y . & l ~ e , B'. A. (1977) Biochem. Biophys. Res. Commun., 78, 631-640.

BIOCHIM1E, 1979, 61, n ° 3.

66. Ya~tfiv, M. ,¢ Folk, ~ . R~. (1~J'7~) J. Biol. Chem., 250, 3243-3253. 67. Rogg, H., Mfiller, P. ~ Staehelin, M. (1975) Eur. J. Biochem., 53, 115-117. 68. Kruppa, J. ~ Zaol~au, I~. G. (1972) Biochim. Biophys. Acta, 277, 499-512. 69. Gef~er, M. L. ~ Russ.el, R. L. (19,69) J. Mol. Biol., 39, 145-157. 70. Dirh~imer, Cr., FA)ell, J. P., B~)n'net, J., Gangloff, J., Keith, C~., Krebs, B~., KuntzO1, B., Roy, A., Weiss'enl~ach, J. a "~x~errmr, C. (1972) Biochimie, 54. 127-144. 71. Dube, S. K. (1:973) FEBS Letters, 36, 39-42. 72. Erdma.nn. V. A., Sprinz], M. ~ Pongs, O. (1973) Biochem. Biophys. Res. Commun., 54, 942-948'. 73. Rich,rex, D., Erdm,ann, V. A. ~ Spr.inzl, M. (19,73) Nature N. B., 246. 132'-135. 74. Schwarz, U., Liihrmann, R. ~ (~assen, H. G. (1974) Biochem. Biophys. Res., Commun., 56, 807-814. 75. Schwarz, U., M,en~e:l, H. X~. ~ Gassen, H. G. (19'76) Biochemistry, 15, 2484-'2~49'0. 76. Schwarz, U. ~ Gassen, H. G. (1977) FEBS Letters, 78, 267-272. 77. Sprinzl, M., VCagn,er, T., Lorenz, S'. ,~ Erdman,n, V. A. (1976) Biochemistry, 15, 3031-3039. 78. Piper, P. W. a Gl,ark, B. F. C. (1973) FEBS Letters, 30, 265-267. 79. Sim, sek, M., Petrissar~t, ~. ~ R a j b h a n d a r y , U. L. (1973) Proc. Nat. Acad. Sci., 70, 2600-2.604. 80. GiH'um, A. M., Hecker, L. I., Si~lberkl~ang, M., S.chwartzbach, S. D., R,ajbh, and,a,ry, U. L. & BarneC~t, W. E. (1977) Nucleic Acids Bes., 4, 41094132. 81. P:iper, P. ~V. ~ Clark, B. F. C. (1974) Eur. J. Biochem., 45, 589-600. 82. Garel, J. P., Garbor, R. L. ~ Siddiqui, M. A. Q. (li977') Biochemistry, 16, 3@18-3624. 83. Gl~en, E. Y. ~ Peo¢, B~. A. (197'8) Biochem. Biophys. Bes. Commun., 82, 235-246. 84. Marcu, K., M,igrmry, R., l~esze.lbach, R., Roe, B., Sirover, M. ~ Dudnck, B. (1973) Biochem. Biophys. Res. Cornmun., 55, 477-483. 81. Randera'th, 5, Ghia, L.,L. S. Y., Morris, H. P. tLanderath, K. (1974~) Biochim. Biophys. Acta, 366, 159-167. 86. P,iper, P. W. (1975) Eur. J. Biochem., 51, 2.95-304. 87. Jank, P., Riesner, D., Gross, It. J. (19'77) Nucleic Acids Res., 4, 20{)9,-291~. 88. Roe, I}. A., Ananda~aj, M. P. J. S., Gh4~a, L. S. Y., Ra~derahh, E., ~ulp[a, R. G. ~ R~a~nderath, K. (19'75~) Bioehem. Biophys. Res. Commun., 66, 1097-1105. 89. R esz~lbach, R:., Green'h~rg, R., Pirtt, R., Prasad, R., Marcu, K. & I)udock, 1~. (1977) Biochim. Biophys. Acta, 475, ~83-392,. 90. D i n s e r m a n n ~, Th,., Schmidt, W. $: Kersten, H. (1977) FEBS Letters, 80, 205-208. 9.1. M,arcu, K. B. ~ Dudock, B. S. (1976) Nature, 261, 159-162. 92, R~e, B. A. z, Tsen, H. Y. (1977) Proc. Nat. Acad. Sci., 74, 3696-3700. 93. Bjork, C~. R. & Neidhardt, F. C. (1975,) J. Bacteriol., 124, 99'-103. 94. Brambilla, R., Rogg, H. • Staehelin, M. (1976) Nature, 263, 167-169. 95. Roberts, J. W. ~ Ca,rbon, J'. (1074) Nature, 250, 412~-41(L 96. lY~ilt,er, J. P., Hussa'in, Z~ ~ S,ehvc~i~er, M. P. (1976) Nucleic Acids Bes., 3, 1185-1201. 97. S(alomon, R , Fuchs, S., A~aroaov, ,~., Giveon, D. IAbtat~e,r, U. Z. (19'75) Biochemistry, 14, 4046-4054. 98. Salomon, R., Giveon, D., K i m h i , - Y . a Littauer, U. Z. (197.6) Biochemistry, 15~ 5258-5262~. 99. Kvit1L G., Rogg, IT., Dirb.~in~r, G., Menichi, B. ITeyman, T. (1976) ~FEBS Letters, 61, 120-123. 100. McLennan, B. D. (1975) Biochem. Biophys. Res. Commun., 65, 345'-351. 101. Feldman, M. Ya~. (19'77) Progr. Biophys. ,~ Mol. Biol., 32, 83-90. 102. McCutch~n, T., S~ilverm,an, S., Ko~'li, J~ a S,611, D. (1975)Biochemistry, 17, 1622-162~8.

tRNA in regulation of biosynthesis. 193. Sclfimmel, P. 1~. (1977) Accounts o f Chem. Res., 1 0 , 411-414. 104. Roe, B., Micka,~l, M. & Dudock, B. (1.973,) Nature, 246, 135-137. 105,. M'unr~s, T. W., Pod~atz, K. C. & Katzman,, P. A. (19,74~) Biochem. Biophys. Res. Commun., 59, 496~501. 106. Munns, T. V¢. & S~im,v, HI. F. (1075) J. Biol. Chem., 250, 2143-2!149. 107. Ch~en, G. S. & S~iddiqui, M'. A. Q. (1975) J. Mol. Biol., 96, 153:-170. 108. Ga,~l)er, R. L., S~iddiqu,i, M. A. Q. & A14man, S. (1978) Proc. Nat. Acad. Sci., 75, 635-639. 109. An~alric, F., Ra~h~eiterie, J~.-P. & Gaboehe, M. (1977) Nucleic Acids Res., 4, 436'7-4~370. 110. Robevtson, I4. D., Alfm~an, S. ~ Sm'ith, J. D. (19'72,) J. Biol. Chem., 247, 5243-5251. 111. KatiE, J. R. (1975') Biochim. Biophys. Aeta, 407, 392~39'8. 112. Oue,HieCte, A. J~. & Taylor, M. W. (19:73) Biochemistry, 12, 3542t-354~6. 113. K ~ r , S. J. ~ B:o~k, E. (1.97;3) i n hl~,e Ado. E n z y m e s Regulation. (W~el~er, G. Ed.,) v.ol. 11, p. 63-77. Per~a.mon Press. 114. Luck, D. N. a Hamilton, T. ~. (19'75) Biochim. Biophys. Acta, 383, 23-29,. 115. K~i,tchingman, G. R. ~ Fourn~ie~r, J. (1977) Biochem i s t r y , 16, 22!13-2220'. 116. Crick, F. H!. G. (196'6) J. Mol. Biol., 19, 548-555. 117. Holmes, V¢. M., Goldm~an, E., M,irrer, T. A. & Ha,tfi~eld, G. V~. (1977) Proc. Nat. Acad. Sci., 74, 139'3-139.7. 118. Mi~tra. S. K. (1'9'78) FI~BS Letters, 91, 78-80. 119. J'ank, P., Sh,indo-Ol~ada, N., N~ishimura, S~. & Gross, L. I~. (1977) Nucleic Acids Res., 4, 1999-2008. 12~D. Mit,ra, S. K., Lusfig, F., Al~esson, R. & Lagevkvist, U. (I977) J. Biol. Chem., 252, 471-478. 121. La~erkvist, U. (197~) Proc. Nat. Acad. Sci., 75, 17,59-1762.

122. Kur~t~el, B., ~re,issenbach, J., W~olf, R. E., Tumaitis-Ke~n,edy, T. D., Larre, R. G. & Dirhedmer, G. (1976) Biochimie, 57, 61-70. 123. Sen, G. C. & Ghosh, H. P. (1976) Nucleic Acids Res., 3, 5,2~%535. 124. Wei,ssen~ba,Ch, J. & Dirheimer, G. (1978) Biochim. Biophys. Acta, 518, 53~)-534. 125. Wong, T V¢., ~ a r r i s , M~. A. & Morris, HI. P'. (1975) Biochem. Biophys. Res. Commun., 65, 1137-1145. 126. F~a~rk~as, W. R. & Ghernoff, D. (1976) Nucleic Acids Res., 3, 252d-2529. 127. Ok~ada, N., H~arad(a, F. & N~isltrimtrra~ S~. (1976) Nucleic Acids Res., 3, 2593-2fi03. 1,28. Okada, N., Y~a~suda, T. a Nishim'u~a, S. (1977) Nucleic Acids Res., 4, 4963-407~. 129. Hilse, K. & Rudl,off, 'E. (1975) FEBS Letters, 66, 38~0~383. 130. E f s t r ~ i a d i s , A., Kafa~tos, E. C. & Martin.is, T. (197'7) Cell, 10, 571-5'86~. 13E V,a.nGall~e~, D. & ~ils~, K. (1974,) FEBS Letters, 39, 56-610. 132. W~it~e, B. N., Dun,n~ R., Gillam~ J~., Tener, G. H., A.rmstrong, D. ~., Skoeg, F., Fr~ha~rt, C2 R. L~ona~d, N'. J~. (1975~ J. Biol. Chem., 250, 515-5~1. 133'. l~at~e~ J. I~. (1975~ Biochim. Biophys. Acta, 383, 131-139. I34. Mc l~eyn.olds, ~,., OZM)alley, B. W., N.i,s,h~, A. I~., F~(~hergil~, J. E., G~vel. D., Fi~l~ds, S., Robertson, M. & B r o w n i ~ , Ct. G. (1978) Nature, 273, 723-72~8. 135. UHrioh, At., Shine, ~., Gkirgwin,, J'., Pictet, R., l~istrer, E., Butter, V¢. ~. ~, Go(~drrran, If. M. (1977) Science, 196, 1313-13r9. 136. Se,~burg, P. I-L, Shine, J., Marti'M, J. A., Baxter, J~. D. & Goodman, ~. M. Nature, 270, 486-7~93. 1'37. Shine., 2., S.eebtrrg, P. I-L, l ~ r t i a l , J. A., Baxter, J. D. & Goodm'a~a, H. M. (1977)Nature, 270, 494499. 138. Fiters, ~V., G(~treras~ R., ~aegen~a'n, G., Rogiers, R., Vande Voorde, A., ~ z n Heuv~rswyn, H., Van I~erreweghe, $., Voleka,ert, G. ,¢ Yset)aert, M. (1978) Nature, 273, 113-12.6.

BIOCH1MIE, 1979, 61~' n ° 3.

341

139. San tier, F., A'ir, G. M., I ~ r r e l l , B. G., l ~ o w n , N. L., Gou,lson, A. R., l~iddes, J. X . HutEhison III, C. A., S~oaombe, P. M. a Smitll, M. (19'77) Nature, 265, 687-692. 140. Fi.ers, W., Con,t~r~ras, R., Duea'inck, F., Ha egeman, G., Iseren.t,ant, D., ~ e r r e g a e r t , J., M~in Jou, W., Molemans, F., Baeym~aeI~.rs, A., Van den Be rghe, A., Votelcaert, G. & Yseba,e~t, M. (1976) Nature, 260, 500-507. 141. M~i~nJou, W., Ha~effeman, G., Ysebaer~, M. & Fievs, W. (1972) Nature, 273, 82-88. 142. Fi.ers, W., Controras, K., D~erin.ck, F., I-Ihegeman, G., M ~ r e g a e r t , J., Min J ou, V¢~., ~aeymake.rs, A., Vo4ckaert, G., Yse~bae~k M., Vain de~ Kerckhove, J., Notf, F. a Van. Mort.tat'u, M. (1975) Nature, 256, 273-278. 143. H~arada, F. a Nfshimura, S. (1974¢) Biochemistry, 13, 3O0-307'. 144. Min J~)u, W., Mon~agu, M. V. & Fiers, W. (1976) Biochem. Biophys. Res. Commun., 73, li083-1093,. 145. Ilan, J~, Ilan, J. a Pa.$d, N.. (1970) J. Biol. Chem., 245, 1275-1281. 1.46. Sll~arm~, O. K., ~tays, L. L. a ]~a~ek, E. (19.73) J. Biol. Chem., 248, 762~2-762¢. 147. Sharma, O. K., M~.ysi L. L. & Rorek, E. (1975) Biochemistry, 14, 5'09~514~. 148. S!haTma, O. K., Blee'~leT, D. N. a R,ol~erts., W. K. (1~976) Biochemistry, 15, 4313-431'8. 149.. V~ainwriglrt, S. & Wa,inw~i~kt, L. K. (1967) Can. J. of Biochem., 45, 265-2!66. 15'0. Wainwrigtrt, S. & Wainw~ighL L. K. (1970) Can. J. of Biochem., 48, 40(~-466. 15L W~ainwrigl~L S. ~ V~ai,nwrigh¢, L. K. (19'72,) Can. J. of Biochem., 50, 1056~10,63. 15~2~ ~r,Mn.wr~ght, S. & Wainw~ig,lR, L. K. (1972) Can. J of Biochem., 50, 1155-1164. 153. F,alk.off, E., Falkoff, P~., Debleu, B. & Revel, M. (1972) Nature N. B., 240, 1~5-147. 154. Samu~d, C. E. a Jo~klfk, W. K. (1974 ~) Virology, 58, 476-~91. 155~. Con/~ent, J., L~bl¢~, B., Zilbe~rsteAn, A., Berissi, H. Rev~l, M. (1974)FEBS Letters, 41, 125-130. 156. Content, J., Lebleu, B., Nud~l, U., Zilbexs'~ein, A., Bierissi, H~. & Rev~l, lye. (19759 Eur. Y. Biochem., 54, 1-10. 157. Gup~a, S. L., Sopori, 55. L. ~ Lerrgu¢,l, P. (197°-) Biochem. Biophgs., Res. Commun., 57, 763-770,. 198. ]~aill, L. A.. a Whti~e, G. N.. (1978`) Proc. Nat. Acad. Sci., 75~ 1167:-1'1ql. 1.59. RaCnew, L~., W i ~ a n d , P~. G., Fia~ell, P. J:., Sen; G. G., Ccabr~r, ~. & :Lengu!~l, P. (1~97'8) Biochem. Biophys. Res. Commun., 81, 947-9,5'4. 1~0. Levin, D. & L o n d o ~ ,L M. (1'978`) Proc. Nat. Acad. Sci., 75, 1421-1125'. 161. LEWIS, J. A., Faleoff, E. & Falcoff, R. (1978) Eur. J. Biochem., 86, 497-509. 162. F~a~lcoff, R., Le~lieu, B% Sanceau, ~., Weis*senbaclL J., D irhe~mer, G~., Ebel, J~. P. & FMcoff, E. (1975) Biochem. Biophys. Res. Commun., 68, 1323~133T. , ~ 1%Icon, ~ v~,,, t Stan 163. W~iss~enl)ach, J., Dirheimev, G., ee~a~t, J. & F~adcoff, E. (1977) FEBS Letters, 82, 71-76 ,. 164. Zilbor~ein., A., Dudock, R., Bea~.issi, H. & R~vel, M. (1~976)~J. Mol. Biol., 108, 43-54. 1~65. M~ay~, U., P~ermaT,e~, ~.-P., W~ei&in~ffer, G., J u n g ~virtJa, C., Gross, I~. ,L & Bodo, G. (1977) Eur. J. Biochem., 76, 541-5,51. 166. Juarez, H., Ju.areZ, D. a H'edgco~th, C. (1974,`) Biochem. Biophys. Res. Commun., 61, 110-116. 167. Dei~on, Y., J~ang, W. K. & S~.rlin, ft. L. (1975) Differentiation, 4, 65-70. 1,68.. Olsen, C. E. a Pen hoe¢, E. E. (1976) B i o c h e m i s t r y , 15, 4649~4654. 169. Richer, L. L. 0978) Biochim. Biophys. Acta, 517, 76 -83~.

170. Piper, P. SV. (1975.) Eur. J. Biochem., 51, 283-293. 171. Hirsh, D. (1070) Nature, 228, 57. 172. Buck~ingham, R.. If. & K url,a~nd, C. G. (1977) Proc. Nat. Acad. Sci., 74, 5496-5498.

342

L. A. Osterman.

173. Rosenf~e.ld, M. (~. ~ t~aa-pile,ux, A. (1977) Biochemistry, 16, 514~518. 174. B l o b d , G. (19737 Proc. Nat. Acad. Sci., 70, 924-9"28. 17'5. Lia.ut,amd, J..-P., S e t y o n o , B., S p i n d l e r , E. & K6.h~ler, K. (197~6) Bioehim. Biophys. Aela, 425, 373:3(83. 176. Huynh-Va.~-'llar~ ¢, Soh~apira, G. (19,78) Etzr. J. Biochem., 85, 2'7'1~-281.

BIOCHIMIE, 1979, 61, n o 3.

177. Li,au,tard~ J.-P. & K6hl~er, K. (1976) Biochimie, 58, 317-323. 1~7~. Spi.rin; A. S. (1978) FEBS Letters, 88, 15-17. 179. K u r l a n d , C. G., R,igler, R., l~hrgnberg, M..~ B lomberg, G. (1975) Proc. Nat. Acad. Sei., 72, 42484251.