the mechanism of translational coupling in e coli

8/6/2019 The Mechanism of Translational Coupling in e Coli

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0 1994 by The American Society for Bioche mistry an d Molecular Biology, Inc.T H E JOURNALF BIOLEICALHEMISTRY Vol. 269, No. 7, Issue of July 8 , pp. 1811&18127,994Printed in U.S.A.

The MechanismofTranslational Coupling n Escherichia coZiHIGHER ORDER STRUCTURE IN THE tpHA mRNA ACTS AS CONFORMATIONAL SWITCH REGULATINGTHE ACCESS OF DE NOVO INITIATING RIBOSOMES*

(Received for publication, January 7 , 1994, nd in revised form, April 26, 1994)Gundula Rex, Brian SurinS, Gerda Besse, Bernard Schneppe, an d Jo hnE.G. McCarthyOFrom the Department of Gene Expression, Gesellschaft fur Biotechnologische Forschung mbH (GBF), Mascheroder Weg 1 ,38124 Braunschweig, Federal Republic of Germany

Bacterial genes are commonly transcribed to formpolycistronic mRNAs bearing reading frames whose re-spective translational efficiencies are not indepen-dently determined. As in many bacterial operons, ex-pression of the at p genes of Escherichia coli is stronglyinfluenced by translational coupling.Thegene pairatpHA is tightly coupled, wherebyatpA is translated atleast three times more efficiently han atpH. However,there is no fixed stoichiometryf coupling: mutations inatpH lead to increases in the translation ratio (atpA/atpH) of up to approximately 40-fold. We have demon-strated that secondary structure sequestering the atpAtranslational initiation region (TIR) s important to thecoupling mechanism in that it nhibits de novo transla-tional initiation at the atpA star t codon. Genetic andstructural analyses indicate that this inhibitory struc-ture can be induced o refold into a less inhibitory con-formation either by introducing two single-basesubsti-tutions or as a result of ribosomes translating atpH. Wepropose a model in which the secondary structure of theatpA TIR acts analogously to a gating device in that itrestricts de novo ribosomal initiation until it isswitched into a more open conformation. This con-trasts with the function of a stem-loop struc ture locatedimmediately downstream of atpA and upstream of theShine-Dalgarno regionof atpG, which was found o in-hibit trans lation, but not to mediate tight coupling. Re-sults obtained using the specialized ribosome systemof Hui and de Boer((1987) roc. Natl. Acad.Sci. U. S. A.84,4762-4766) ndicate tha t primarily ribosomes reiniti-ating after termination on atpH are responsible for in-ducing refolding of the atpA TIR. The principle of alter-native mRNA conformations with different functionalproperties embodied in the model presented here canonlybefulfilledby certain types of structure. It islikely to operate in several steps of prokaryotic geneexpression, underlying a range of regulatory eventsincluding transcriptional attenuation and translationalactivation.

The ability of RNA to form secondary and ter tiary structu reis essential to its multifarious functions in living organisms.Yet very little is known about the funct ional significance ofspecific RNA struc tures n cellu lar processes. An importantexample of the role of higher orderRNA struct ure is to be found* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must thereforee hereby markedaduertisement n accordance with 18 U.S.C. Section 1734 solely toindicate this fact.$ Present address: CSIRO Divisionof Plant Industry,GPO Bo x 1600,Canberra, ACT 2601,Australia.0 To whom correspondence shouldbe addressed. Tel.:0531-6181-430;Fax: 0531-6181-458.

in he control of trans lation. Both ineukaryotesand pro-karyotes, the efficiency of translational initiation can be con-trolled by secondary struc tures in t he mRNA tha t effectivelyrestric t the accessibility of the initiat ion site 1 , 2). At least atth e phenomenological level, inves tigations of a range of sys-tems have pointed to the importanceof both the stability andthe position of stem-loop structures in the leade r or in thevicinity of the start codon of various mRNAs, although a gen-erally applicable universal definition of th e region(s) of anmRNA molecule likely to be relevan t to the ontrol of transla-tional initiation still remains tantalizingly beyond our grasp(2-5). In th e iving cell, higher order RNA structure is respon-sible for variations in trans lational initia tion efficiency over arelative range stretching from the highest rates appro achingthe max imum possible loading and elongation rates of ribo-somes, down t o rates apparently restrictedby at least a factorof 1000.

The accessibility of prokaryotic t ransl ational initia tion re-gions (TIRs, defined in Ref. 2) t o ribosomes can be restricted inthe steady state by means of secondary structure. Indeed, atleast in the caseof the bacteriophage MS2 coat protein gene,the predicted thermodynamic stability f a defined stem-loop inthe TIR correlates well with the apparent level of restrictionexercised on transl ation in Escherichia coli (6). However, aspointed out elsewhere 71 , beyond this typeof thermodynamiccontrol, kinet ic control may also be exercised by the TIR atspecific steps of the initia tion pathway (8, 9). Moreover, t hekinetics of formation (and perhapsof disruption) of the stem-loop structures themselves may exert an important influenceon th e role of secondary structure. Indeed, one mRNA speciesmay support the formation of a number of alternative struc-tures, whereby each may have a different rate of formation.There is little known about the interchangeability of such al-ternative structures. In general, we are a long way from un-dersta nding the roles of the kinetics and thermodynamics offormation of higher order structure in te rmsof the function ofindividual domainsor complete molecules of mRNA.

The present work is concerned with an im portan t, ye t ftenneglected aspec t of translat ional control in prokaryotes. A argeproportion of all bacterial genes are transcribed o form poly-cistronic mRNAs bearing eading rames whose respectivetrans lational efficiencies ar e n o t independently determined. In-deed, many genes are translationallycoupled, a phenomenonthat is as widespread as it is poorly understood (2, 10-24).Translational coupling also seems t o occur in eukaryotic or-ganelles (251, and reinitiation may play an importa nt role inthe ranslational regulation of some eukaryoticgenes (26).Again, at least in th e prokaryotic systems, higher order struc-ture in the mRNA often plays an important mediating role.

Theabbreviations used are: TIR, translational initiation region;ASD, anti-Shine-Dalgarno.18118


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Mechanism of Translational Coupling in the E . coli atp Operon 18119The a tp operon of E. coli is an xample of a polycistronic mRNAwhose function is strongly influenced by translational coupling(21, 27-31). The coupling is intrinsic o hegeneration ofamounts of the component subunits of the membrane-associ-ated proton-translocating ATP synthase (H+-ATPase) hat ar ematched to thetoichiometry of this enzyme complex (cy3&y16,

a, b, clccls).The order f the genes seems to bearo functionalsignificance obviously related to theespective rates of synthe-sis of the subunits, but isikely t o be relevant to theassemblypathway of the H+-ATPase (32, 33) .

Aparticularly striking xample of coupling is provided by thegene paira t p H A (Fig. 1). In this ase, the d ownstreamcoupledgene a t p A is translated at least three times more efficientlythan the gene ( a t p H ) o which it is coupled (18) . Prokaryoticribosomes are known to reinit iate fficiently under appropriateconditions (13, 15), and this provides a n explanation for thecoupling of many closely juxtaposed bacterial genes (e .g . theribosomal protein genes 34)) .Yet reinitiation alone cannot ex-plain the stoichiometry of coupling of th e a t p H A gene pair. Afacilitated binding mechanism,nvolving the melting f a stem-loop structure in the t p A TIR, ha s been proposed as an alte r-native pathway (18) . This type of mechanism is also likely tounderl ie the oupling between the ribosomal subunit genes L10and L7/L12, where the synthesis rate of the la tter is at leastfour times higher than that f the former (10, 13) .

We have addresseda question tha t is f general relevance tothe function of mRNA structure in a wide range of biologicalprocesses. What is the mechanism of involvement of the pro-posed mRNA structure, and what ole is played by the dynam-ics of its folding and unfolding? We have applied geneticas wellas biochemical analyses to study the RNA struc ture involvedin a t p H A coupling. The interaction etween the struc tured tp ATIR a nd ribosomes ha s been investigated using mutationalanalysis as well as a novel approach based on the use of spe-cialized ribosomes whose mutatedanti-Shine-Dalgarno se-quence directs them to initiate atorrespondingly altered Shine-Dalgarno sequences (35 , 36). We have come to the conclusionth at a switch n the onformation of th e mRNA is intrinsic othe coupling mechanism. We present a model in which the struc-ture initially formed in the a t p A TIR acts analogously t o agating device that inhibi ts translat ion until it is forced t orefold into a more open conformation by a ribosome translatingthe upstream a tpH reading frame. Transient structuresf thetype described here may frequently play important regulatoryroles in both trans lation and otherrocesses in the ell. Finally,we compare the structural and functional properties of th ea t p H A structure with those of a stem-loop stru ctur e in theintercistronic region between a t p A and a tpG which is found t ofulfill a different role in the control of a tp gene expression.

EXPERIMENTAL PROCEDURESSA 500 his ilu b8lacZXA21 (A int2 xis1 nutL3 c1857A H I ) .

Bacterial Strains-The bacterial host was the E. coli strain A1563:Plasm id Constructions-The la c2 fusion vector and the general re-

combinant DNA methods used weres described previously (18,21). norder to facilitate co nstruction of further fusions involving atpA andlacZ, in vitro mutagenesis was used to eliminate an EcoRI site in theN-terminal equence of atpA,creat ing A B CAA CTC AAT TCC,whereby the underlin edATG is the sta rtodon of atpA, and the furth erunderlined position (now C)s a G in the original sequence.his modi-fication does not change h e encoded amino acid sequenceof atpA andis in fact located in loop region of the mRNA struc ture shown in ig.3. The atpHA::lacZ construct referred to in this pape r always containsthe described mutation.

atpA(AEcoRIIC1aI)::lac.Z:atpHA::lacZ was first cleaved with EcoRIin the olylinker of the expression vector (upst ream of the inse rted atpsequence; see Ref. 18) and with ClaI (position 3098 in the atp operon(see 37)). The ends were filled-in and religated.

atpH(ATC)A::lacZ: th e ATG start codon of atpH inatpHA::lacZ waschang ed to ATC by in vitro mutagenesis.

atpH(ABstEII1 C1aI)A::lac.Z: atpHA::lacZ was pa rt ially cleaved withBstEII (atp operon 2781) and ClaI (atp operon 3098) and ligated with asynthetic oligonucleotide which connected both ends in frame.atpH(ABstEII/ClaI,O)A::lacZ nd + I 1-1 derivatives: atpHA::lacZwas partially cleaved with BstEII (atp operon 2781) and ClaI (atpoperon 3098)and ligated with syntheticligonucleotides which carr ieda Shine-Dalgarno sequence and a startodon in thewild-type readingframe of atpH (0) r in the+1 o r -1 reading frames, respectively.atpFHA::lacZ: Hp aI an d NarI cleavages of atpFH::lacZ (18) werefollowed by a fill-in reaction. The resulting fragment was ligated withatpHA::lacZ which had been cut withEcoRI and subsequently treatedwith Klenow enzyme.

atpH(hBstEII1 C1aI)::lac.Z: at pH (B st EI I1C1aI)A::lacZ was cleavedwith AflII. The single-stranded ends including the TAA stop codonofatpH were removed by m eans of mung-bean nuclease digestion. Thevector was then cut with HindI II and ligated with a synthetic oligo-nucleotide which connected he atpH(ABstEIIlC1aI) g ene and the ac2gene in the correct reading frame.atpH(hBstEIIlClaI,O)::lacZatpH(ABstEIIlClaI,O)A::lacZ wascutwith ClaI, and he resulting vector was igated with he ClaYClaIfragment of atpH(ABstEIIIC1aI)::lac.Z carrying the atpH::lacZ fusionsegment.

atpH(ATC)::lacZ: atpH(ATC)A::lacZ was cleaved with ClaI, and theresultin g vector was igatedwith heClaVClaI ragment of atpH-(hBstEIIlC1aI)::lac.Z.atpH(ASD,ABstEIIlClaI)A::lacZ: a BamHI site was introduced intoatpH(hBstEIIlC1aI)A::lac.Z between the Shine-Dalgarno sequence andthe sta rt codon of atp H using in vitro mutagenesis. Subsequently, theresulting plasmid was cut with EcoRI and BamH I to remove the 5-untranslated region ofatpH and ligated with a syntheticligonucleotidewhich carried a CCTCC anti-Shine-Dalgarno sequence instead of thewild-type sequence GGAGG.atpH(hBstEIIlClaI)A(ASD)::lacZ ive nucleotides of atpH(ABstEII1C1aI)A::lacZ res idin g in the 1 7th a nd 1 9thodon of atpA were changedby in vitro mutagenesis in order to preventmRNA secondary structurealterations once an anti-Shine-Dalgarno sequence was introduced up-stre am of atpA. The resulting plasmid was thenleaved with AflII an dSphI and ligate d with synthetic oligonucleotide which introduced heanti-Shine-Dalgarno sequence CCCCACTCCTC ins tead of th e nat ura lShine-Dalgarno sequenceGGGGACTGGAG upstream of atpA.atpH(ASD, ABstEII 1ClaI)A(ASD)::lacZ atpH(ASD, ABstEIIl C1aI)A::atpH(ABstEIIlClaI)A(ASD)::lacZ.lac2 was cut with ClaI and ligated with the ClaYClaI fragmentrom

pBIlrrn BASDIX a plasmid was constructed ha t directed synthesisof 16S rRNAin which the SD sequence CCUCC nea r the endof th errnB gene had been mutatedo GGAGG (referred t o as ASDM in ef.35).The mutated rrn3 gene was inserted into the 3amHI site behindthe p,ac promoter of th e expression vector pBI (21). This vector is aderivative of pACYC184 (38), nd herefore ompatiblewith heatp::lacZ expression vectors described above.

In Vitro Danscription Templates-The pGEM construct pGEM-4.317) carried the bacteriophage T7 promoterollowed by a DNA frag-ment bearing the end of atpH and the end of atpA. pBJC1888 (39)was cleaved by ClaI (position 30981, whereu pon the ends were filled- in,and the vector was cleaved again using XhoI (position 3417). The re-sulting 317 bp fragment was ligated withGEM4 (40) which had beencleaved with HindIII, treated with lenow enzyme, and cleaved againwit h Sal I. oth the wild-type atp region 3098-3417 and the mut4 equiv-alent were inserted (yieldingpGEM4.317 and pGEM4.317mut4).

RNA Str uctura l Analysis-The DNA templa te pGEM4.317 (orpGEM4.317mut4) was linearized eithery the restriction enzyme om-bination NaeIiXbaI, o r the combination NaeVBcLI, and trans cribe d invitro using T7 RNA polymerase according to the manufacturers (Pro-mega) specifications. The transcription products were analyzedy aga-rose gel electrophoresis. NaeI cleaves once in th e vector. The oth ercleavages allowed the generation of transcripts of lengths 317 nucleo-tides (XbaI) or 150 nucleotides (BclI),espectively. Both were used forstructural analysis of the atpHA coupling region . The ends of the invitro transcripts were labeled using [a-32PlGTP and guanylyl transfer-ase or [y-32PlATP and polynucleotide kinase 41 ).The adiolabeledRNAs were purified usinga Sephadex G-25 column and used in RNasedigestion assays. These assays were routinely performedt 30 C in 10f ~ l ~ris-HC1, pH 7.0,10 mM MgCl,, 100 mM KC1 with 0.2 pg pl-l E. colitRNA as carrier, as described previously (42), except where otherwisestated (see Fig. 4).


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Mechanism of Tkanslational Coupling in the E. coli atp Operonreading frames -1 and +1 would have been expected t o beterminated at internal stop codons within the atpH readingframe downstream of the ClaI site. However, these internalout-of-frame stop codons are closely followed by n-frame ATGcodons preceded by potential Shine-Dalgarno sequences tha tpresumably allow coupling o in-frame translation of atpH, ashas been described previously for the l a d gene (22). Again,atpH::lacZ fusions were generated by deleting the atpA sectionbetween AflII and HindIII. The p-galactosidase activities sup-ported by these latterconstructs confirmed th at deletion of themiddle section of atpH ( a t p H ( ~ s t E I I l C 1 a I ) : : l a c Z )rasticallyreduces internal initiation in this gene and that int erna l ini-tiation is restored by the introduction of a synthetic initiationsite (atpH(ABstEIIlClaI,O)::lacZ).

In the natu ral polycistronic atp mRNA, atpH translation iscoupled to translation of atpF. Again, translation of atpH islikely to be restricted by secondary structure in the atp HTIRunless at pF is translated 21). Comparison of the P-galactosid-ase levels supported by atpHA::lacZ and atpFHA::lacZ (Fig. 1,Table I) reveals that the nhanced translation of atpH affordedby coupling t o at pF brings about an increase in atpA transla-tion, al though th is "carry-over" effect is relatively small com-pared with the enhancement in initiationat the atpHIR (seeatpFH::lacZ).

It should be emphasized that reliable interpretation of theenzymic activity data presented in thispaper (Table I) can onlybe achieved in the ight of control experiments tha t determinethe relative amounts of the P-galactosidase fusionproteins andmRNAs encoded by the respective l a c 2 constructs. As in pre-vious studies (see e.g. (18, 21)), we found th at the relativeamounts of atp::lacZ mRNA varied by no more than 50% fromone construct t o another (data not shown). Moreover, he rela-tive rates of synthesis of the respective fusion proteins corre-lated directly with the P-galactosidase activities measured.Thus, we conclude tha t the elative values presented in Table Ifaithfully reflect variations in the rateof translation.In conclusion, our data indicate tha t the translational cou-pling of atpA to atp H in the asic construct atpHA::lacZ is to alarge extentdriven by (relatively inefficient) nternal initiationin atpH. The translation of atpA is restricted by the mRNAstructure lying downstream of the ClaI site n atp H (seeatpA(hEcoRIlC1aI)::lacZ) unless ribosomes initiate at henatural atpH start codon or within the atp H reading frame,whereupon the atpA TIR functions much more efficiently.Thecoupling of atpH translation t o atpF also leads to increasedtranslation of atpA, most probably due to an increased rate ofinitiation at thecorrect st ar t codon of atpH.

The Stoichiometry of Coupling of atpHA Can Vary Over aWide Range-Whatever the mechanism of initiation of ribo-somes in the atpH reading frame, the results reported aboveindicate that the oupling between atp H and atpA is not subjectto a strict stoichiometric relationship. This becomes especiallyclear when the apparent relative rates of translational initia-tion for atpA are plotted against t he corresponding rates foratpH (Fig. 2). The ratio of P-galactosidase activities for compa-rable atpHA::lacZ and atpH::lacZ fusions can vary from ap-proximately 5 to more than 200. Moreover, it is clear tha t arelatively poor rate of atpH translation suffices to promote ahigh rate of atpA translation. The curve is skewed toward ahighly amplified response of atpA translation to relativelysmall increases in atpH translation up to a plateau, whereapparently amaximal level of atpA translation is reached (Fig.2). This behavior contrasts strongly with t he theoretically ex-pected linear relationship of a gene pair that is ranslationally

a i18121

00.0 0.1 0.2 0.3 0.4atpH translation rate

FIG. . T he apparent stoichiometry of coupling between atpHand atpA is variable. A graphical comparison of the relative /3-galac-tosidase activity levels encoded by atpHA::lacZ fusions compared withthe equivalent atpH::lacZ fusions. Plotted are thevalues for the a t p HAclones atpH(ABstEIIlClaI)A::lacZ,atpH(ATC)A::lacZ,tpHA::lacZ,atpH(ABstEIIlClaI,O)A::lacZ,nd atpFHA::lacZ against the values forthe correspondingatpH::lacZ clones ( in which atpA' has been deleted).Increasing the very low rate of atpH translation in atpHA::lacZ bymeans of mutagenesis results in greatly amplified increases in thetranslational rate of atpA. The rate of translational initiation on a t p Atha t is apparently driven by coupling o translation of atpH levels of f ata maximum value. The data displayed here indicate that the ratio oftranslation (a tpH:atpA)can vary over a wide range, reaching a maxi-mum value of approximately 1200.

The Roleof Secondary Structure in the Coupling MechanismofatpHA-Previous computer-assisted analyses of the possiblesecondary structure formed in and around the natural atpHAintercistronic region predicted the existence of a structure atleast similar t o that shown in Fig. 3 (18, 21). Mutational anal-ysis was consistent with the idea that secondary structureplays a role in the coupling mechanism (21). In one mutant,referred to as atpHA::lacZmut4, two adjacent A+ U mutationsare sufficient t o allow the atpA(AEcoRI1C1aI)::lacZ construct(Fig. l), hich is normally very poorly translated, to be trans-lated approximately 15 imes more efficiently.t should be notedthat in this mutant theutations reduce the complementarityof the upstream Shine-Dalgarno region (nearest to the atpHstop codon) to the ribosomal 16 S rRNA anti-Shine-Dalgarnoregion. On the other hand, computer-assisted analysis (43) pre-dicts that themut4 mRNA will assume a structure different tothat formed by the natu ral tpHA intercistronic region (Fig.3).We tested the hypothesis tha t the nat ura ltpHA mRNA forms(a t least initially) a structure of the type illustrated in Fig. 3,but tha t an alternative structure is favored when the G+ Umutations of mut4 are introduced into the sequence.

RNase analysis was performed using in vitro transcriptsbearing the wild-type and mut4 derivative of the atpHA mRNA,including the intercistronic region and the surrounding se-quences believed to participate in the formation of inhibitorysecondary structure (Fig. 3). A range of RNase concentrationswas used in order to establish which of the bands resultingfrom structural analysis were attributable to single cleavagesof otherwise complete RNA molecules (Fig. 4). A series of ex-periments was performed in order to characterize the influenceof variables in the ncubation conditions, such as Mg2' concen-tration andfolding temperature of the RNA, upon the cleavagepatterns. The results observed with both the 317 and 150 nucle-otide RNA species using the single strand and double strandspecific RNases are consistent with the existence of the pre-dicted structures (Fig. 3). The structure predicted using 115nucleotides of the naturalatpHA shown n Fig. 3 fits the RNasecleavage data better than an alternative structurepredicted onthe basis of computer analysis of only 101 nucleotides in thisregion (21). The structure predicted for th e nu t4 equence wasalso supported by the results obtained through RNase treat-ment. It should be emphasized that the stability of the struc-coupledia a fixed stoichiometric mechanism. ture predicted using an RNA folding prediction program (43)


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Mechanism of Dansla t ionaloupl ingnhe E. coli atpperon 18123

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F I G. . mRNA s t r u c t u r a l a n a l y s i sn vitro. Examples are shownof the dat a used to map the single-stranded and double-stranded regionsfthe wild-typea t p H A mRNA. Results are hown of experiments performed with the longer317 nucleotides. A and shorter 150 nucleotides. R andC ) ragme nts that oth include thea t p H A coupling region. Changes in the distribution and intensityf bands as a function of RNase Concentration(given in enzyme units/pg target NA) could be usedas a guide toward optimal cleavage conditions, underhich the greatzv part f the populationhad been cleaved only once per molecule (seee.g. th e T2 anes in A and R) .The VI l anes in A and R as wel l as theT2 a n m In C show the resultsof assays thatwere performed at th is mi nimal RNaseevel. The positions of the cleavages were determined by comparing the hand patternsw t hsequencing reactions performed using concentrationsf RNases A and Iat which all of the bonds that cane specifically r eco plz ed hy these twoenzymes were hydrolyzed. The cleavage data sets obtained using the in vitro transcrip ts derived from the 31 7 and 150 templates were fullyconsistent with each other (compare A


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18124 Mechanism of Tkanslational Coupling in the E . coli at p O peron

atpH(ASD,ABstErI/CkI)A(ASD)::kcz

ASD SD

SD ASD

ASD ASD

B ASD - sequence 5bf atpH: A A A C T A G A C T A A A C A A T A G A A T A T T C A A m ] G A T C C A > T C TC SD - sequence 5 ~ a t p ~ : C T G T A A G G A G m ] G G C T G u T C T3199D ASD - sequence 5'ofatpA: ~ C T T A C C C C A C T ~ C A T G C A L...... C A T T G G A G A G T G G A A T ~E SD - sequence 5bf atpA: T C T U J , G G G G A C T [ G ~ C A G C A A A / . . . . . . . ' C A T T G C T C A G T T C A A T G /

322226 127 73 1 9 7 3 2 2 226 127 7

w -IUI Sphlin which the wild-type Shine-Dalgarno regions of either atpH, a tpA,or of both genes, were altered to anti-Shine-Dalgarno( A S D ) egions ( A ) .TheFIG. . Constructs used to direct translational initiationy specialized ribosomes.Three derivatives of atpHA::lacZ were constructedwild-type and synthetic ( A S D )non-coding sequences upstream of atpH (B and C ) and of a t p A ( D nd E ) are shown.TABLE1f ianslational activi t ies of A S D m u t a n t sof atpHA::lacZ

atp::lacZ fusions" Relative P-galactosidaseactivitiesbatpA(Aflll,ASD)::lacZatpH(ABstEII 1ClaI)A(ASD)::lacZ 0.0 (0.0)1.0 2.4)'atpH(ASD,ABstEIIlClaI)A(ASD)::lacZatpH(ASD,ABstEIIlClaI)A::lacZ 0.44 (0.33)5.2 (6.1)These are the ASD constructs depicted in Fig. 5 .These are mean values, each derived fromat least three independ-ent measurements using cell extracts from cells carrying the specifiedconstructs.The values are given for extracts derived from cells in the absenceof, or after (in brackets) induction of pBUrrnBASDIX by IPTG. Allvalues were normalized to the P-galactosidase activity specified by a t -pA(AflII,ASD)::lacZ (without IFTG induction). The absolute activity ofthe latter (here set t o 1.0 as a reference value) was 10 times lower thanthat of atpA(AflII)::lacZ(see text and Ref. 18). n the complete absenceof ASD-specific ribosomes i .e . where pBUrrnBASDM was not presentin the cells), none of the atpA(ASD)::lacZ fusions directed P-galactosid-ase activity (data not shown).

tosidase despite the fact tha t the atpH and atpA TIRs con-tained, respectively, ASD and wild-type Shine-Dalgarno se-quences. However, in this case there was appreciable activityeven without the presence of specialized ribosomes. This wasprobably due t o the fact thathe atpH(ASD) TIR inatpH(ASD,hBstEIIlClaI) A::lacZ was modified using a syn-thetic non-translated sequence that disrupted the wild-typesecondary structure in this region. Thus, although there is notypical Shine-Dalgarno sequence in the atpH(ASD) TIR of thisclone, it is an -rich, relatively open stretch of mRNA contain-ing sequences such as UAA and UAAG that might promote alow level of binding of wild-type ribosomes. As we have seen(Fig. 21, this would be expected to result in ameasurable levelof coupled translation of atpA. We therefore conclude that thecoupled translation of atpA::lacZ in atpH(ASD,ABstEIIl

C1aI)A::lacZ is primarily due to translation of atpH by wild-type ribosomes, and therefore provides us with little informa-tion about the ability ofASD ribosomes to facilitate theinitiation of wild-type ribosomes in the atpA TIR. The resultsobtained with the other ASD constructs, on the other hand,indicate that reinitiation of ribosomes that have translatedatpH plays a major role in themechanism of coupling to trans-lational initiation in the atpA TIR.The Stem-loopStructure between atpA and atpG Plays aFunctionally Distinct Role in Danslational Control-There isalso stable secondary structure in the region downstream ofatpA (see structure prediction in Fig. 3). Particularly striking isthe large stem-loop immediately downstream of the atpA read-ing frame, in the 50-nucleotide long intercistronic region be-tween atpA and atpG. Since earlier data (21) indicated thatthere is translational coupling between atpA and atpG, weasked the question as t o whether this secondary structuremight fulfill the same role as tha t formed in the atpHA inter-cistronic region. Previous work (44) howed that mutations inthe atpG TIR th at reduced the stability of predicted intramo-lecular secondary structure allowed greatly increased transla-tional initiation at the tp G start codon. However, hese muta-tions changed nucleotides downstream of the Shine-Dalgarnosequence, within the atpG reading frame, or in both of theseregions. In the present investigation, we introduced mutationsspecifically designed t o destabilize the stem-loop whose stemincludes two nucleotides of the atpA stop codon at it s base (seeatpAGmut::lacZ in Table I11 and Fig. 3). These mutations allowgreatly increased translation of atpG, thus confirming that thisstem-loop strongly inhibits translational initiation. However,given that it isot responsible for tight coupling between tpAand atpG (44),t evidently functions differently from the struc-ture involved in the coupling mechanism of atpHA.


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Mechanism of lFanslationa1oupling in the E . coli at p Operon 18125TABLE11lkanslational activitiesof atpAG::lacZ constructs

atp::lacZ fusions Relative P-galactosidaseactivitiesbatpAG::lacZ 1.00atpAGmut::lacZ 15.3

These are the atpAG constructs depicted in Fig. 3.Mean values calculated as described in Table I normalized to thevalue for atpAG::lacZ.

DISCUSSIONWe have established that the oupled translation of atpA can

be driven not only by ribosomes initiating at the atpH startcodon, but also by those th at have initiated internally withinthe reading frame f this gene. There is no fixed stoichiometricrelationship between the respective translational rates of atpHand atpA; the ratio can vary by up to a factor 40 as a functionof the a tpH translation rate, whereby atpA translation appar-ently does not exceed a certa in maximal value. Moreover, nei-ther initiation at the atpH start codon nor synthesis of thefull-length atpH product (subunit 6) is a prerequisite of tra ns-lational coupling. This conclusion is fully consistent with theformer observation that the expression of atpH in trans doesnot mediate coupling to atpA (21). Thus, the previous historyof ribosomes transla ting theC-terminal region of a tpH seems t obe irrelevant t o the mechanism of coupling. However,at leastunder normal physiological conditions in vivo, the majority ofribosomes terminating the atpH reading frame in the polycis-tronic at p mRNA willhave initiated at the ona fide star t codonof this gene (see later).

Previous discussions of the possible mechanisms responsiblefor translational coupling have focused on two general ways inwhich ribosomes might par ticipate (2, 10-24). In the first ofthese , ribosomes terminating transla tion at th e stop codon ofthe upstream gene (in this case a t p H ) subsequently re initiateat the st ar t odon of the downstream gene (in this case atpA).It is believed tha t the ribosomes dissociate after termination,but tha t at east the 30 S ribosomal subunit scans along themRNA until it locates the sta rtcodon of the downstream gene,whereupon the 70 S ribosome reassembles. This pathway doesnot involve de novo binding of 30 S ribosomal subunits, andthus allows maximally one initiation event at thedownstreamstar t codon per termination at th e upstream stop codon. Thesecond type of mechanism depends on the ability of translatingribosomes to disrupt intramolecular higher order structure inthe mRNA. The destabilization of such structure in the down-stream TIR (in this case tha t of atpA) renders the Shine-Dal-garno region and star t codon of this gene accessible to 30 Sribosomal subunits whose binding would otherwise be pre-vented. This facilitated binding mechanism is a t least partlycatalytic, in the sense that ribosomes translating atpH acti-vate a TIR whose ribosomal binding site is otherwise seques-tered by higher order structure. Only this lat ter ype of mecha-nism can explain how an apparently tightly coupled gene suchas atpA can be translated much more efficiently han the genelying upstream of it on th e mRNA ( a t p H ) .Neither of the abovetwo ways in which ribosomes participate in coupling excludesthe other. Coupling is of course only possible ifhe downstreamcoupled gene does not possess an efficiently functioningand/or freely accessible TIR.Thus, the coupling either removesa structural barrier to initiation (as in the case of atpHA) orallows re initiation at a TIR which contains only a poorly rec-ognizable Shine-Dalgarno region and/or a suboptimal startcodon (15,161. However, the above considerations alone are notsufficient to provide an explanation of the variability of thecoupling ratio observed with atpHA (Fig. 2).

n

1l20

on the principle of facilitated bindinp of ribosomes toanFIG.6. Alternative pathways of translational couplingbased

otherwise inaccessible binding ite. The TIR of a tpA assumes, atleast initially, a conformation tha t inhibits trans lational initiation. Thesecondary structure is disrupted by a ribosome approaching,or bindingto , the stop codon ofa tp H . The disruption may be caused by a ribosomethat reinitiates after terminating on a tpH (pa thways 3 and 4 ) . Alter-natively, a terminating ribosome may disrupt the structure withouthaving t o reinitiate , although reinitiation can occur (p a th w a ys1 and2).The secondary structure might be able to reform after disruption, thu sundergoing a cycle of disruption and refolding, the frequency of whichwould depend on the translational ra te of a tp H . This cyclical type ofmechanism could be riggered by ribosomes tha t either reinitiate p a t h -way 3), o r by ribosomes that disrupt the structure of the a tpA TIR asthey approach the end of atpH and terminate without necessarily hav-ing to reinitiate (p a th w a y I ) . Translation of a tpA would only occurduring the phase of the cycle where the structure was in theunfoldedstate. Both pathways 1 and 3 would be expected to impose a certainratio (within limits of statistical fluctuation) on the coupling process.Alternatively, a non-cyclical type of mechanism might apply, in whichthe inhibitory structure is disrupted only once, either by a reinitiatingribosome (p a th w a y 41, or by a ribosome terminating on atpH (anddisassociating from the mRNA p a th w a y 2).This lat ter type of mecha-ble, whereby he a tp A TIR remains in the open, permissive onforma-nism therefore involves a singular disruption event which is irreversi-tion, being translated by de novo initiating (and reinitiating) ribosomesuntil i t is degraded. The open conformation f the a tpA TIR depictedhere is likely to involve a t least partial formation of an alternativestructure with a stability similar to that of the inhibitory structure (seeFig. 6) . The mut4 mRNAseems to formhis alternative ype of structure(Fig. 3) spontaneously, possibly for kinetic reasons . One o r other of thevarious pathways considered here, or combinations of them, may applyt o many of the translationally coupled genes in bacteria. In the presentwork, we conclude th at pathway 4 most closely describes the mecha-nism of translational coupling in the case of a tpHA.

We have confirmed that the structure predicted (21) for thecoupling region of the atpHA mRNA can form (Figs. 3 and 4).Given tha t thestability of this structure (predicted to be -14.9kcal mol at 37 C (43)) would be expected to inhibit veryeffectively independent translational initiation at thestartcodon of atpA, we are obliged to incorporate it into any modelfor the translationalcontrol of atpA (Fig.6). ll of the pathwaysdepicted in Fig. 6 predict that this structure is destabilized(disrupted) by a ribosome approaching, o r binding to, the stop


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18126 Mechanism of Translational Coupling in the E . coli at p O peroncodon of a t p H . However, in one type of model (pathways 3 an d41, th is destabilization is brought about exclusively by ribo-somes terminating on a tpH that then reinitiate n a t p A . In theother case, destabilizationis induced by a ribosome completingth e a tpH polypeptide chain , but s not solely dependent onreinitiation ( p a t h w a y s 1 and 2 ) ,although reinitiaion can ccur.Whichever of these two routes leads t o destabilization of thehairpin structure, its disruption might occur as part of a re-peated cycle of events ( p a t h w a y s 1 and 3 ) , nvolving sequentia ldisruption and eformation of the structu re asfunction of thefrequency of translation of a t p H . Translation of a t p A wouldonly occur during the phase of the cycle where the stem-loopstructure was disrupted. Thisyclical type of mechanism wouldspecify a res tric ted range of possible stoichiometries of cou-pling. In the alt ernativ e, non-cyclical type of mechanism, dis-ruption of the stem-loop occurs only once ( p a t h w a y s 2 and 4),rendering subsequent initiations at thetp A start codon inde-pendent of a tpH translation. This latter typef pathway there-fore constitutes a type of switching, in which an inhibitoryconformation of the a t p A TIR is forced to refold into a lessinhib itory struc ture which is of comparable stability. The lessinhibitory structure allows independent ranslational nitia-tion which is no longer tightly coupled t o the trans lation ofa t p H .

The results obtained here using he specialized ribosomesystem ndicate that reinitiation plays a major role in hemechanism of translational coupling between a tpH and a t p A( p a t h w a y 3 o r 4 n Fig. 6). I t therefore does not seemsufficientfor ribosomes merely t o translate, or bindo, parts of the a tpHreading frame; they must terminate trans lation at the a tpHstop codon and reinitiate at th e a t p A start codon in order topromote efficient transla tion of a t p A . Moreover, the variabilityof th e stoichiometry of coupling (Fig. 1,which allows enormousdiscrepancies between the espective ra te s of trans lation, leadsus to onclude that a cyclical disruption and reformationf thestem-loop stru ctur e in the t p A TIR is unlikely. Overall, theseconsiderations point to pathway 4 (Fig. 6) as being nearest tothe operative mechanismof translational coupling. There aretwo potential Shine-Dalgarno regions in the intercistronic re-gion of a t p H A , whereby one of these, which comprises twothree-nucleotide sequences with continuous complementarityto the 16 rRNA, is directly adjacent to the stopodon of a tpH(Fig. 3). The second begins two nucleotides downstream of thefirst and endsonly one nucleotide upstream of the a t p A startcodon. The combination of the two Shine-Dalgarno regionsmight be predicted to facilitate the transferf the terminatingribosome to form a n initiation complex at th e a t p A sta rt codon,although the econd one is in position that would normally bejudged non-optimal for translation. The bindingof a reinitiat-ing ribosome to the second Shine-Dalgarno region may there-fore contribute in a more indirect way t o the coupling process.I t is unlikely that th e stem-loop structure is imply opened upby the reiniti ating ribosome, leaving the TIR relatively un-structured. We suggest that themore likely pathway involvesdestabilization of the original stem-loop and refolding of themRNA to form a struc ture of the type determined for the mut4mutant form of the a t p A TIR (Fig. 7) . This seems feasible inview of t he fact tha t the alternative struc ture has a similarpredicted stability to that formed initially by the wild-typemRNA sequence. Bothstructures are stablen v i t ro ,whereas invivo the mRNA is switched from one conformation (the inhibi -tory stem-loop) o the other (the lessnhibitory, mut4-like struc-ture). However, we do not know for what proportion of themRNAs lifetime subsequent to conformational switch ing thealternative structure isully o r partly formed. This will dependon the kineticsf ribosomal ini tiat ion and longation, as well as

ribosometstop

15

FIG. . Alternative mRNA conformation model for atpHA. Inthis model, the structure initially formed by the atpA TIR is disruptedby a ribosome that terminates on atpH and reinitiates on @A. ThemRNA is able to refold as a consequence of the interaction with thereinitiating ribosome, formingan alternative structure that is at leastsimilar to the structure formed by the mut4 mRNA in uitro. This alter-native structure renders the Shine-Dalgarno regions and the s tar tcodon of a tpA accessible tode nouo initiating ribosomes. Oncede nouoinitiation at the atpA st ar t codon has begun, the mRNA doesnot readilyrevert to the initial, inhibitory structure (on the left-hand side).Whether the alternative structure can fully refold between initiationevents is unknown. Therefore, this model functions analogously to aconformational switch or gating mechanism.on the kinetics of (re-)folding of the mRNA.

Comparison with thea tpAG gene pair reveals that the pres-ence of an inhibitory stem-loop in the region where couplingcan take place is no guarantee that two genes will be (tightly)trans lationally coupled. The large tpA-proximal stem-loop vi-dently allows ribosomes t o initiate at the a tpG star t codon atapproximately half he normal rate even in he absence oftranslation of a t p A (21, 44). Destabilizing this stem-loop in-creases ranslation markedly (Table 111). It was also shownpreviously th at anmRNA fragment bearin g thetp G TIR plusflanking sequences binds 30 S ribosomal subunits with verylow affinity (45).We think it likely th at while restricting trans-lational initiation at th e a tpG start codon, its unfolding byribosomes that translate a t p A , whether partial or complete, isof limited significance in terms of coupling, partly because ofthe length of the a tpAG intercistronic region (compare (17)).Alternatively, the stem-loop (perhaps in a partially unwoundform) may be sufficiently stable to prevent scanningf ribo-somes that have terminated on a t p A . In conclusion, th e func-tion of a stem-loop in terms of translational control and cou-pling is evidently dependent in complex way on its stru ctureand position relative to therecognition signals responsible fortermination and initiation.

The physiological significance of the mechan ism roposed fortranslational coupling of a t p H A could relate to the .assemblypathw ay for th e H+-ATPase. The order of the a tp genes (ZBE-F H A G D C ) is such that theomponents of the membrane-inte-grated, proton-translocating F, portion of th e H+-ATPase ar eencoded by the promoter-proximal genes a tpBEF. The solubleF, par t of the enzyme, which catalyzes ATP synthesis whenbound to the membrane-integrated F, complex, is encoded bythe genes a tpHAGDC. This gene order is also maint ained inequivalent operons in other bacteria, but the genes are notalways organized into a single operon (46-53). Neverthe less , inall cases studiedo far, the genes ncoding the 6 and a subunitsar e contiguous. The productsof a t p A G D , the subunitsa, , andp, respectively, have been found o form a complex with ATPaseactivity (54). The binding of this subunit complex to the F,complex is mediated by subunits 6 and E (encoded by a tpH anda t p C , respectively). Subunit 6 is the functional counterpart fthe oligomycin sensitivity-conferring protein of mitochondria(55). It is believed to play an essential role at the interfacebetween theF, and F, components of the H+-ATPase (56) and to


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Mechanism of 57-anslational Couplingn the E . coli atp Operon 18127be involved in the assembly of a functional F, complex (32,57-59). The transla tional coupling mechanism described inthis paper might ensure that ynthesis of subunit a! is switchedon at the arliest shortly before translation of the atpH readingframe is complete, thus possibly ensuring tha t no a! subunitsare synthesized before 6 subunits become available, which canthen in turn elp mediate binding to the F, complex. Althoughinternal initiationwithin atpH can allow coupled ranslation ofatpA, this will be of much reduced significance where atpH islocated in its normal position downstream of atpF in the poly-cistronic operon, since here synthesis of the full-length Spolypeptide will dominate (compare Ref. 60). Clearly, the sig-nificance of this controlled order of synthesis would be greaterwhere synthesis i s closely regulated in a (membrane-associ-ated) microenvironment in the immediate vicinity of eachmolecule of polycistronicatp mRNA. The synthesis of subunit 6is also coupled to the translation of atpF, which encodes the bsubunit of the F, complex (21 ,27,2 9,32) .These considerationssuggest that there may be couplingbetween de novo synthesisof subunits and their ssembly into an active membrane-boundcomplex.

The switching mechanism presented in Fig. 7 is reminiscentof other regulatory mechanisms involving alternative confor-mations of the mRNA. The common theme is that ribosomesinfluence the choice as to which of the alternative structuresis formed, whereby one of the alternatives has a specific func-tion (e.g. to inhibit translation, or to act as a transcriptionalterminator). Examples include attenuation (611, translationalinhibition involving S15 (621, as well as translational ctivationof the bacteriophage A cIIIgene (63) and of the ermC TIR (64).Not only the stability, but also the respective rates of formation(and perhaps f disruption) can be expected to determine whichof the alternative structures are favored. Thus, the inhibitorystem-loop structure in theatpA TIR may be formed primarilybecause it is kinetically favored. The alternative mut4 type ofstructure (Fig. 7) is of similar stability, but may be only rela-tively slow t o form after transcription of this region of the atpoperon. The region of the atp mRNA containing the wild-typestructure may therefore fulfill the function of a gating mecha-nism, which needs to be switched to the active state by ribo-somes before atpA can be translated. In a wider context, theeffectively irreversible type of conformational switching ofpathways 2 and 4 in Fig. 6 may frequently represent the un-derlying mechanism of translational coupling between pro-karyotic genes. Further detailed genetic and biochemicalanalyses will be required to thoroughly test this hypothesis.

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