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RNA Biology

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Is the cellular initiation of translation an exclusiveproperty of the initiator tRNAs?

Sunil Shetty, Souvik Bhattacharyya & Umesh Varshney

To cite this article: Sunil Shetty, Souvik Bhattacharyya & Umesh Varshney (2015) Is the cellularinitiation of translation an exclusive property of the initiator tRNAs?, RNA Biology, 12:7,675-680, DOI: 10.1080/15476286.2015.1043507

To link to this article: http://dx.doi.org/10.1080/15476286.2015.1043507

Accepted author version posted online: 21May 2015.Published online: 21 May 2015.

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Is the cellular initiation of translation an exclusive propertyof the initiator tRNAs?

Sunil Shetty1, Souvik Bhattacharyya1, and Umesh Varshney1,2,*1Department of Microbiology and Cell Biology; Indian Institute of Science; Bangalore, India; 2Jawaharlal Nehru Center for Advanced Scientific Research;

Jakkur, Bangalore, India

Translation of mRNAs is the primaryfunction of the ribosomal machin-

ery. Although cells allow for a certainlevel of translational errors/mistransla-tion (which may well be a strategic need),maintenance of the fidelity of translationis vital for the cellular function and fit-ness. The P-site bound initiator tRNAselects the start codon in an mRNA andspecifies the reading frame. A direct P-site binding of the initiator tRNA is afunction of its special structural features,ribosomal elements, and the initiationfactors. A highly conserved feature of the3 consecutive G:C base pairs (3GC pairs)in the anticodon stem of the initiatortRNAs is vital in directing it to the P-site. Mutations in the 3GC pairs dimin-ish/abolish initiation under normal phys-iological conditions. Using moleculargenetics approaches, we have identifiedconditions that allow initiation with themutant tRNAs in Escherichia coli. Dur-ing our studies, we have uncovered anovel phenomenon of in vivo initiationby elongator tRNAs. Here, we recapitu-late how the cellular abundance of theinitiator tRNA, and nucleoside modifica-tions in rRNA are connected with thetRNA selection in the P-site. We thendiscuss our recent finding of how a con-served feature in the mRNA, the Shine-Dalgarno sequence, influences tRNAselection in the P-site.

Introduction

Translation of an mRNA is a highlyregulated process. It is the cellular mecha-nism to generate proteome diversity with-out affecting the basic repertoire of the

genetic information. Prokaryotes devotemore than 30% of their dry mass and~95% of the total RNA for the translationmachinery itself to ensure accurate andefficient translation.1,2 Translation ofmRNA occurs in the steps of initiation,elongation and termination followed byribosome recycling for another round ofinitiation. Initiation is thought to be themost rate-limiting step in translation.3 Ineubacteria, the canonical pathway of initi-ation begins with the formation of a 30Spre-initiation complex (PIC) involvingthe small subunit of the ribosome (30S),mRNA, initiation factors (IF1, IF2 andIF3) and the initiator tRNA (tRNAfMet).The tRNAfMet plays an important role inselecting the start codon. Any failures atthis step could lead to mRNA translationin incorrect reading frames and produc-tion of mis-translated peptides. Dockingof the large subunit of the ribosome (50S)converts the 30S PIC into the 70S initia-tion complex (IC). In both complexes,tRNAfMet is present in a tilted conforma-tion between the P/E and P/P state, the P/I state.4,5 Upon GTP hydrolysis (medi-ated by IF2), the ribosome undergoestransitions, which locate tRNAfMet in theP/P state and result in release of the initia-tion factors (Fig. 1). At this point the 70Scomplex is ready to accept elongatortRNAs in its A-site.5 The selection oftRNAfMet in the P-site is assisted by theribosome bound initiation factors, the ele-ments in the P-site and the distinctstructural features in tRNAfMet.6-17 Wehave been interested in understandingthe mechanism of initiation from a‘tRNAfMet-centric’ view using an in vivoassay system we developed nearly 3 deca-des ago.18 In this short article, we

Keywords: anti-Shine-Dalgarno, aSD, ini-tiation with elongator tRNAs, proteomediversity/plasticity, ribosomal heterogene-ity, Shine-Dalgarno, SD, 3GC base pairs,70S initiation

*Correspondence to: Umesh Varshney; Email:varshney@mcbl.iisc.ernet.in; uvarshney@gmail.com

Submitted: 03/12/2015

Revised: 04/16/2015

Accepted: 04/17/2015

http://dx.doi.org/10.1080/15476286.2015.1043507

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RNA Biology 12:7, 675--680; July 2015; © 2015 Taylor & Francis Group, LLCPOINT-OF-VIEW

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summarize our findings toward under-standing the mechanism of initiation in E.coli. To lead up to these findings, we firstdiscuss the salient structural features of theinitiator tRNA and the in vivo assay sys-tem central to our studies.

Initiator tRNA: StructuralFeatures and Cross Talk with

the Ribosomal Elements

The prokaryotic tRNAfMet are charac-terized by at least 3 special features

(Fig. 2). Firstly, they possess a non-Wat-son-Crick pair (C1 £ A72 in E. coli) atthe top of the acceptor stem. This featureis an important determinant for, (a) for-mylation of the amino acid attached totRNAfMet, (b) avoidance of binding of theaminoacylated tRNAfMet to EF-Tu, and(c) prevention of hydrolysis of the formyl-aminoacyl-tRNAfMet by peptidyl-tRNAhydrolase (Pth), an essential enzymewhich recycles tRNAs from the peptidyl-tRNAs that fall off the translating ribo-somes.17,19,20 Secondly, at position 11:24(in DHU stem), tRNAfMet possesses apurine:pyrimidine pair in contrast to theother tRNAs, which possess a pyrimidine:purine pair at this position. A knownfunction of this feature is to contribute tothe formylation efficiency of the tRNA.19

Thirdly, a feature that is highly conservedin initiator tRNAs from all domains oflife, is the presence of 3 consecutive G:Cbase pairs (GC/GC/GC or 3GC pairs) inthe anticodon stem at positions 29:41,30:40 and 31:39. The primary function ofthis feature is to facilitate preferentialbinding of the initiator tRNA in the ribo-somal P-site.21-23

Of the 3 distinctive features intRNAfMet, 2 i. e., the presence of a non-Watson-Crick pair at the top of the accep-tor stem and the 3GC pairs in the antico-don stem are the most crucial. Whenincorporated into an elongator tRNA,they allow it to function as an initiator.24

The property of formylation of tRNAfMet

increases its affinity to IF2.22,25 However,unlike formylation which occurs only ineubacteria, 3GC pairs are almost

Figure 1. Translation initiation in eubacteria. The formation of 30S pre-initiation complex (PIC) involves binding of all the 3 initiation factors IF1, IF2 andIF3 along with tRNAfMet and mRNA. The tRNAfMet in this stage is slightly tilted away from the P-site, called P/I state. This complex recruits 50S subunitwith the help of IF2 to form 70S IC with the tRNAfMet in P/I state. This IC undergoes a rotational movement upon GTP hydrolysis to give rise to a 70S com-plex with the tRNAfMet in P/P state. This rotation also leads to release of initiation factors from the 70S to convert into an elongation competent 70S.

Figure 2. Cloverleaf structure of E. coli tRNAfMet2 (metY). Sequences important for its special proper-ties are shown in colored box. CxA mismatch at 1£72 position (shown in orange color) togetherwith the base pairs at 2:71, 3:70 is a major determinant for formylation of aminoacid attached totRNAfMet. The 11:24 position (shown in yellow) also affects the efficiency of formylation. The 3 con-secutive GC base pairs in anticodon stem (in green box) are important for the ribosomal P- site tar-geting of the initiator tRNA.

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universally conserved in the initiatortRNAs emphasizing their functional sig-nificance.21,26 Mutational analyses haveshown that of the 3GC pairs, the middleGC pair is the most critical one. Interest-ingly, even though tRNAfMet retainingjust the middle GC pair (and mimickingthe unusual tRNAfMet from some of themycoplasmas or rhizobia27) functions ininitiation, the naturally occurring elonga-tor tRNAs with even 2 GC base pairs(including the middle GC pair) do not.Thus, it appears that the context of the3GC pairs vis a vis rest of the tRNA mayalso be important. However, it should alsobe said that at least in yeast, the require-ment of the 3GC pairs in the initiatortRNA seems more flexible as changing themiddle GC pair to CG pair could still sus-tain its growth.28 These observations sug-gest that the precise mechanistic role ofthe 3GC pairs in initiation is still an openquestion. How the 3GC pairs help tRNAf-

Met in its function of P-site binding/initia-tion has been the focus of our studies.

The ribosome bound initiation fac-tors,6-8 the elements in the P-site such asthe 16S rRNA nucleotides G1338 andA1339 (known to interact with the 3GC

pairs)29,30; methylated nucleotidesm2G966 and m5C96731,32 and the tails ofthe ribosomal proteins S9 and S1331,33,34

etc. are also known to contribute totRNAfMet selection in the P-site. Besides,there is evidence that methylations atother positions in 16S rRNA play a role intRNAfMet selection.35-37 The possibilitythat the rRNA methylations impact theaffinity between the 50S and 30S subu-nits38 needs further investigation. It hasbeen suggested that a rapid rate of 50Ssubunit docking onto the 30S PIC mayadversely impact the fidelity of tRNAfMet

selection in the initiation complex.7,8

In vivo Assay System, Mutationsin the 3GC Base Pairs in tRNAfMet

and Isolation of Suppressors

Our in vivo assay is a plasmid-based sys-tem, which employs chloramphenicol(Cm) acetyltransferase (CAT) reporterwhere the AUG start codon has beenmutated to an amber codon (UAG). Thisreporter (CATam1) mRNA fails to conferCm resistance to E. coli because the cellulartRNAfMet (with CAU anticodon) does not

initiate from the ‘UAG’ start codon(Fig. 3). However, when the cells are sup-plied with the tRNAfMet whose anticodon ismutated to CUA (from CAU), initiationfrom the UAG start codon produces CATprotein conferring CmR to the cell.18,22,39

The tRNAfMet with CUA anticodon is ami-noacylated with Gln by GlnRS.18 Whenthe 3GC pairs in the anticodon stem of thetRNAfMet are mutated to those found in theelongator tRNAMet (UA/CG/AC, called3GC mutant tRNAfMet), the mutant tRNAloses its competence to initiate even thoughit is efficiently aminoacylated and formy-lated, rendering the host Cm sensitive(CmS). We used this phenotype of CmS ofthe host in a suppressor analysis to charac-terize mutations in the E. coli chromosome[introduced by N-methyl-N’-nitro-N-nitrosoguanidine, (MNNG) treatment]that conferred CmR to the host byenabling the 3GC mutant tRNAfMet toinitiate from the CATam1 reporter.36

Identification of the chromosomalmutations in the suppressor strains(Fig. 3) discussed below resulted incharacterization of mechanisms thatcontribute to tRNAfMet selection in theP-site, and in the fidelity of initiation.

Figure 3. In vivo assay system to investigate the role of 3GC pairs in tRNAfMet. The plasmid based assay system employs CATam1 reporter with amber startcodon together with a tRNAfMet containing CUA anticodon (derived frommetYCUA gene). ThemetYCUA encoded tRNA is aminoacylated with Gln and thenformylated by 10-formyltetrahydrofolate:L-methionyl-tRNAfMetN-formyltransferase (Fmt)18 to produce fGln-tRNAfMet,CUA. The UAG start codon of CATam1

is recognized by metYCUA encoded tRNA to produce CAT protein, which confers chloramphenicol resistance (CmR) to the cell (A). When the 3GC basepairs in the metYCUA encoded tRNA are changed to UA/CG/AC pairs (3GC mutation), the mutant tRNA does not function in initiation (even though it isefficiently aminoacylated and formylated) rendering the cell CmS (B). Extragenic mutations that rescue the initiation defect of the 3GC mutant tRNA allowinitiation to occur from the UAG start codon of CATam1 mRNA to produce CT and confer CmR (C).

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Contribution of Cellular InitiatortRNA Levels

In E. coli, tRNAfMet is encoded by 2loci, metZWV at 63.5 min and metY at71.5 min. The metZWV locus possesses 3genes in an operon encoding tRNAfMet1

and the metY locus possesses a singletRNAfMet gene (as a first gene of themetY-nusA-infB operon) encoding tRNAf-

Met2.40 In E. coli B strains, both the lociencode identical tRNAfMet.41 However, inE. coli K strains, the metY encoded tRNAf-

Met differs in that it possesses an A (insteadof m7G) at position 4641 with no apparentfunctional differences. The metZWVencoded tRNAfMet contributes to about75% of the total tRNAfMet in E. coli. Inan earlier work, one class of the suppressorstrains which fostered initiation with the3GC mutant tRNAfMet, we observedmutations in the metZWV promoterwhich led to a severe down-regulation ofthe chromosomally encoded tRNAfMet.42

Further, deletion of the metZWV locusalso allowed initiation with the 3GCmutant tRNAfMet suggesting that 3GCpairs offer a competitive advantage totRNAfMet to bind the P-site. In theDmetZWV strain we observed that notonly the unusual tRNAfMet of the myco-plasma and rhizobia origin (lacking eitherthe first, the third or both the first and thethird GC pairs) but also the elongatortRNAs initiated protein synthesis.42,43 Infact, initiation by the unusual tRNAfMet

sustained growth of E. coli in the absenceof all 4 of the tRNAfMet genes.27 Normallythese unusual tRNAs fail to initiate in E.coli to any significant level. Thus,although a higher number of initiatortRNA genes has been attributed to over-come the rate limiting step of initiation,our studies suggest that higher cellularabundance of tRNAfMet makes a definitecontribution to the fidelity of initiation bypreventing initiation with elongatortRNAs. The relative levels of tRNAfMet

and elongator tRNAs and their aminoacy-lated forms are known to vary understress.44-47 At least in E. coli, ppGpp aswell as cAMP-CAP regulate transcriptionof tRNAfMet genes.48,44 Thus, it is possi-ble that under the stressful conditions,initiation (from noncanonical positions)by elongator tRNAs generates novel

polypeptides by translation in alternate orthe original reading frames. Physiologicalrelevance of any such novel polypeptidesis unknown. However, the potential toinitiate with elongator tRNAs may serveas a cellular strategy to generate proteomediversity which in turn may rescue cellsfrom stress. Interesting as the observationof in vivo initiation with elongator tRNAsis, it raises several questions. How arethese tRNAs recruited to the ribosome?Also, as initiation with elongator tRNAs iscompeted out by tRNAfMet, the elongatorsmust initiate from the P-site. If so, is therelative affinity of tRNAs for EF-Tu andIF2 somehow influenced? Could an elon-gator tRNA (in its EF-Tu bound form)reposition itself into the P-site for initia-tion? Given that E. coli can grow in theabsence of formylation49 and that an EF-Tu bound aminoacyl-tRNA may be re-sampled50 by other proteins in cell,answers to these questions may not beimprobable.

Contribution of Shine-Dalgarno(SD): anti-SD (aSD) Interaction

Recently, we observed that in yetanother suppressor strain, a mutation(C1536T) in the aSD sequence of a 16SrRNA gene which resulted in an extendedpairing of 8 (from the pre-existing 6) basepairs with the SD sequence of the CATam1

mRNA enabled initiation with the 3GCmutant tRNAfMet.51 Similarly, a mutationin the SD sequence of the CATam1

reporter mRNA resulting in its extendedinteraction of 8 base pairs in the sameregion of aSD of the wild-type 16S rRNAalso resulted in initiation with the 3GCmutant tRNAfMet. While the exact mecha-nism remains to be investigated, weobserved that the extended SD:aSD inter-action resulted in an increased occupancyof the 3GC mutant tRNAfMet onto theelongation competent 70S ribosomes sug-gesting that at least one of the roles of the3GC pairs in the native tRNAfMet is tofacilitate its transition from 30S PIC tothe 70S complex. The 3GC pairs may be‘licensing’ the tRNAfMet to pass throughthe multistep process of the scrutiny byinitiation factors (e. g. IF36,7,29) and/orthe conformational changes the ribosome

undergoes (e. g. upon GTP hydrolysis5) toyield an elongation competent 70S(Fig. 1). A systematic mutational analysisof the 3GC base pairs in tRNAfMet mightoffer further insights into these processes.For example, in our earlier studies, weshowed that overproduction of the 3GCmutant tRNAfMet could also lead to aminor increase in initiation, an observa-tion consistent with the role of 3GC basepairs in the direct binding of the tRNAf-

Met to the 30S ribosome.42,43 It is alsoknown that an extended SD stabilizes the70S complex.52,53 Consistent with this,we observed that kasugamycin treatment(which is known to allow 70S mode ofinitiation in leaderless mRNAs54–56)offered a selective advantage to initiationwith the 3GC mutant tRNAfMet from theCATam1 mRNA with the extended SDsequence. Other conditions such asincreased IF2 levels or decreased RRFactivity or decreased IF3 activity (condi-tions that increase 70S population in cell)also facilitate initiation with the 3GCmutant tRNAfMet. These observationssuggest that the requirement of the 3GCpairs is more acute for the canonical modeof initiation from 30S ribosome. Thewild-type tRNAfMet is known to facilitaterecruitment of a leaderless mRNA fortranslation in the 70S mode of initia-tion.57 Conversely, could a stably boundmRNA in 70S ribosome facilitate P-siterecruitment of a tRNA via codon:antico-don interaction?

G1338 and A1339, 2 of the highlyconserved residues of 16S rRNA, havebeen shown to form type II and type I A-minor interactions with the first and themiddle GC base pairs of the 3 GC pairs,respectively.33 However, as the absence ofany of the GC pairs affects the efficiencyof initiation, it may well be that duringthe various stages (transitions) of the initi-ation process, G1338/A1339 makedynamic interactions with all the 3 of theGC base pairs to offer discriminationbetween tRNAfMet and the elongatortRNAs.29,58,59 Thus, any displacements inthe locations of G1338 and A1339 mayimpact the selection/retention of a tRNAin the P-site. In this context, it has beensuggested60 that the h26 and h28 in 30Smove apart by »2A

�upon interaction

between SD and aSD sequences resulting

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in a displacement of G1338 and A1339 by~13 A

�compared with their location in the

empty ribosomes (without mRNA ortRNA). In the absence of availability ofribosome structures that directly addressthese changes, it is unclear how such dif-ferences might impact the scrutiny of the3GC pairs. However, among the 3 initia-tion factors, IF3 plays an important rolein selection of 3GC pairs6,61 throughG1338 and A1339.29 A displacement ofthese residues due to an extended SD:aSDinteraction might attenuate the IF3 medi-ated scrutiny of the 3GC pairs, and allowinitiation with 3GC mutant tRNAs. Atleast, from the perspective of the geneticanalyses, an extended SD:aSD interactionseems to compensate for the conforma-tional changes that might be broughtabout by the interaction of the G1338and A1339 with the 3GC pairs of thetRNAfMet to allow initiation to proceedeven in the absence of the 3GC pairs.

Outlook

Using 3GC mutant tRNAfMet in ourgenetic analyses, we have explored cellularmechanisms that contribute to the mainte-nance of fidelity of translation initiation,especially in the selection of tRNAfMet inthe P-site. Interestingly, these studies havealso uncovered cellular strategies that gener-ate heterogeneity in the ribosomal pool36,38

(for example, a combinatorial effect of lesserthan quantitative modifications of theknown nucleoside positions would generatean enormous diversity/heterogeneity ofarrangements of rRNA modifications avail-able in the individual ribosomes), whichmay allow selective/preferential translationof mRNAs in cell to generate proteomediversity/plasticity. Earlier studies haveshown heterogeneity in the ribosomal poolthat arose as a consequence of various envi-ronmental cues or changes in the toxin-anti-toxin systems.62,63 Cellular abundance ofinitiator tRNA has been thought to beimportant in regulating the rate-limitingstep of initiation. Revelation that the poten-tial of elongator tRNAs to initiate isunleashed by decreasing pools of initiatortRNA, and given that relative levels of initi-ator/elongator tRNAs in the cell dochange,48 it seems that alternative sites of

initiation determined by the elongatortRNAs may generate novel polypeptides tocreate another level of proteome diversity/plasticity in the cell. Such diversity may becrucial for the cell to re-program cellularphysiology to tide over the stressful condi-tions. There are circumstances where initia-tor tRNA levels are transcriptionally down-regulated44,48 or diminished by ribonu-cleases such as VapC.64 Furthermore,although not yet experimentally tested, ourrecent finding that extended SD:aSD inter-action allows for initiation with an initiatortRNA lacking the highly conserved featureof the 3GC pairs suggests that the naturaloccurrences of SD sequences in translationinitiation regions or at internal positionscould also impact the extent and/or sites ofinitiation by elongator tRNAs. It is knownthat there are various mRNAs withextended SD sequences in the upstream orwithin the coding regions which lead topausing of ribosomes.65 Preference for SDsequence uses has already been shown tovary with temperature.66 It is also of interestto note that the 70S ribosomes from E. coliwith low levels of tRNAfMet contained 15%less S1 protein.43 Earlier studies have shownthat S1 depleted ribosomes use 70S modeof initiation in leaderless mRNAs.55,57

Thus, it seems that cells may exploit a num-ber of strategies available to it to generateproteome diversity. However, a challengethat these observations pose is to designmethods to detect and identify minor popu-lations of peptides/proteins, which are tolikely arise by initiation from the alternativesites. Clearly, more sensitive genetic andbiochemical assays would need to be devel-oped to investigate into the possibility ofthe impact of the alternative means of initia-tion by elongator tRNAs.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest weredisclosed.

Funding

The work in the investigator’s laboratoryis supported by funds from the Departmentof Science and Technology (DST), Depart-ment of Biotechnology (DBT), and theCouncil of Scientific and IndustrialResearch (CSIR), New Delhi. U.V. is a J.

C. Bose fellow of DST. S.S. is supported bya Shyama Prasad Mukherjee SRF of CSIR.S.B. was supported by an SRF of CSIR.

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