A target site for translational regulation

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Journal Club A target site for translational

regulation Translattonal regulauon has long been recogmzed as a mechamsm for modulating gene expression m orgamsms rangmg from bacteriophage to vertebrates The avadab~hty of m R N A for antmuon of protem synthests can be a functmn of the secondary (or ternary) structure adopted by the R N A molecule. Selectwe bmdmg by regulatory protems may alter m R N A s t r u c t u r e t o affect t r a n s l a u o n a l e ~ c m n c y

or snnply block tmuatmn Perhaps among the best studied bacterial systems which exlablt translaUonal regulation are the nbosomal protem operons of E cob (see Ref 1 for a review) The synthesis of some of the ribosomal protems is coor&nated and, m some cases, sto,chmmetncally balanced Some of the operons have been shown to be regulated by a translaoonal feedback mechanism m wbJch one of the polypeptide products select,vely bmds the mRNA to prevent further synthesm of some or all of the operon's protems 1 Recent efforts have centered on structural eharacterizauon of these mRNA target sttes An extenswely studied system is the L l l operon. Recently, Nomura and las colleagues have used a random mutagenesm approach to answer questions about the role of mRNA structure m translational regulatmn of tl~s operon 2 The LI1 ribosomal protem operon, named for the operon's first astron, consists of the LI 1 and L1 clstronsL Translation of ribosomal protein L1 is coupled to that of the precedmg L l l A number of m v~vo and m vitro studies have shown that LI regulates operon express,on at the translatmnal level by specdicaily binding to an mRNA region located near, or at, the translatmn imtmtaon s,te of Lll. According to the current model, L1 recogmzes a s,te on the mRNA that structurally resembles the L1 binding site on 23S nbosomal RNA (rRNA)L3 Although its affimty for 23S rRNA ts greater than that for =ts mRNA, L1 wdl brad its m R N A when 23S rRNA is hnutmg In tins way, synthests of L1 and L l l ]s coordinated with the avadabd,ty of 23S rRHA as mature nbosomes are assembled In support of tlus compeutmn

model, L1 mbJbmon of translauon of the operon can be relieved m vitro by additton of 23S rRNA 4. _Also, Nomura and Ins colleagues have used deleuon analys,s to tmphcate a pomon of the leader region of L1 l/L1 mRNA m translatmnal feedback control5 Exammauon of L1 bmdmg s~tes on 23S rRNAs from various orgamsms has revealed a common presumptwe secondary structure In one E coh 23S rRNA model, for which there is some expenmental evidence6, nucleoudes 2093-

2196 fold into a rather long doublestranded stem punctuated by loops and bulges 7,s. Stem and loop btructures for the translatmn ,muaUon region of L l l / LI m R N A can be drawn to resemble a phylogeneUcally conserved subsectmn of the E coh L1 bmdmg stte23.8. To test for structural homology between these two RNAs and to define the L1 target me m the L l l / L I leader mRNA, Thomas and Nomura designed a random mutagenesm procedure to ~solate and subsequently characterize a senes of point mutauom m the mRNA that no longer allow translauonal regulatton by L1 (Ref 2) Plasm~ds bearing the e n ~ e LI1/L1 leader regton plus the first 12 codons of the LI l gene fused m phase to/acZ were subjected to UV mutagenem Because A

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mP.NA(R~2, 6-8) D~eregzonofpo~te~ homology as blocke~ St~s of pomt m : ~ o n s m the mRNA whwhlughl~ht th~ homology am mda3~edm bold leben: The presumpu~ Slune-Lc~gamo sequence (G-A-G-G-A) is indicated by ~ m the mRNA structure 71mj ~ r e t s adapted from Figures 2a and 2b ofRef 2

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~) 1987 ~Pubhcauons Cambridge 0376-5067/87,'$02(]0

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T I B S 1 2 - November 1987

the construmon puts the Lll-[3-galactos~dase fusion under translauonal control by L1, mutants were screened for [3galactosldase acuwty on mchcator plates Selected mutants were checked for [3galactosldase acuvlty m a strata that overproduces L1 (and, therefore, greatly represses synthesis of the unmutated fusion protem) and in a strata winch is wdd type for L1 synthesis The rallo of actmty m the latter versus the former strata indicates the nature of the mutatlou; iugh ratios reflect little or no interference with L1 regulation whereas low raUos md~cate that the mutauon exerts a strong neganve effect on the regulauon Thomas and Nomura found that mutatmns winch greatly repress, or abohsh, regulation generally cluster around nucleoudes 48-54 and 74-76 of the Lll/L1 leader m R N A These two regions are part of a 'stem-bulge-stem' structure m the LII/L1 m R N A model that may be structurally homologous to the aforementioned phylogenetlcally conserved subsection of the L1 binding site m E colt 23S r R N A A comparison of the two structures, based on data reported by Thomas and Nomura 2, is shown schemaucally in Fig 1 The regmn of possible structural homology is boxed and sites of point mutations m the m R N A which hlghhght it are indicated m bold letters Thomas and Nomura suggest that the mare determinants for LI recognmon may be the bulge-loop separating two short GC-nch stem structures (Fig. 1). L1 may interact directly ruth tlac bulge smce mutation of bulge residue A53 to any other base abohshes regulation The GC-rich stems may serve an essential

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structural role because mutations whuch the molecular basis for specific protemperturb tlus base pmrmg destroy or R N A mteractmns that regulate translaseverely hmlt regulation This notion ns tion supported further by earher work of Nomura and his colleagues which References I Nomura. M, Gourse, R and Baughman, G dc~'umented the importance of the stem (1984) Annu Rev Bwchem 53,75-117 structure mvoPong nucleot~des 49-51 2 Thomas,M S and Nomura,M (1987)Nuclew and 74-76, but not of its pr~nary Aculs Res 15, 3085-3096 sequence, for repression by L1 (Ref. 9) 3 Nomura, M, Yates,J L, Dean, D and Post, Overall, the data reported by Thomas L (1980) Proc NaiIAcad Sct USA 81,5389and Nomura further support the concept 5393 that structural smulant~es between the 4 Yates, J L and Nomura M (1981) Cell 24, 243--249 m R N A binding site and 23S r R N A brad5 Baughman,G andNomura, M (1983)Ce1134, mg s3te for L1 account for translaUonal 979--988 regulation Compentuon between the 6 Noller, H F 0984)Annu Rev Biochern 53, two bnnchng sntes would modulate the 119-162 translation of L l l and LI m R N A to 7 Gourse, R L, Thurlow, D L, Gerbn, S A match the synthesis of r R N A m the cell and Ztmmetman, R A (1981)Proc Nail The importance of the conserved bulgeAcad Sa USA 78, 2722-2726 8 Branlant,C, Korl, A , Machatt.A and Ebel, stem region m E coh 23S r R N A can also J-P (1981)Nucleic Acids Res 9,293--307 be ascertmned experwaentally 9 Baughman, G and Nomura, M (1984) Proc These stuches on the nnteractmn beNatl Aead Sct USA 81,5389-5393 tween L1 and the L l l / L I m R N A illus- 10 Carey.J, Lowary,P T and Uhlenbeck,O C trate the Importance of R N A secondary (1983) B~ochenustry 22, 4723-4730 structure m some protein-nucleic a o d 11 Romamuk,P J and Uhlenbeck,O C (1985) &ochenastry 24, 4239-.4244 mteracuons in a second example of translauonal regulauon, Uhlenbeck and 12 Rornamuk, P J, Lowary, P, Wu, H-N, Stonno, G and Uhlenbeck. O C (1987) co-workers have used various synthebc Btochenustry 26, 1563--1568 ohgonbonucleotides to study the physi13 Springer,M, Plumbndge, J A, Buder, J S cal interaction between R17 phage coat Graffee, M, Dondon, J. Mayaux,J F, Fayat, protein and its site of translational repG, LesUenne,T. Blanquet, S and Gmnbergression on R17 RNA (Refs 10-12). Manago.M (1985)3 Mo/ Bio/ 185,93--104 Another example of negative autogen- 14 Spnnger, M, Graffee, M, Butler, J S and Gmnberg-Manago,M (1986)Proc NaiIAcad ous translational control is that seen w~th So USA 83, 4384-4388 the thrS gene of E colt Here, threonyl 15 Butler, J S, Spnnger, M, Dondon, J and t R N A synthetase presumably binds a Grunberg-Manago,M (1986)./ Bactenol 165, 5'untranslated region of its own m R N A 198-203 that shares both primary and secondary structure analogies vdth specific parts of RUTHM STARZYK several threomne isoacceptor tRNAs (Refs 13-15) Future work on these Departmentof Biology,MassachusettsInstituteof three systems should further elucidate Technology.Cambridge,MA 02/39, USA ,