Affinity labelling in situ of the bL12 protein on E coli 70Sribosomes by means of a tRNA dialdehyde derivative

Received March 22 2017 accepted June 29 2017 published online August 16 2017

Codjo Hountondji1 Jean-Bernard Crechet2Jean-Pierre Le Caer3 Veronique Lancelot1Jean AH Cognet4 and Soria Baouz1

1Sorbonne Universites UPMC Univ Paris 06 Unite de RechercheUPMC UR6 lsquolsquoEnzymologie de lrsquoARNrsquorsquo 4 Place Jussieu F-75252Paris Cedex 05 France 2Ecole Polytechnique Route de SaclayF-91120 Palaiseau France 3Institut de Chimie des SubstancesNaturelles CNRS-ICSN UPR2301 Universite Paris-Sud Avenuede la Terrasse F-91198 Gif-sur-Yvette France and 4LaboratoireJean Perrin Sorbonne Universites UPMC Univ Paris 06 UMRCNRS UPMC 8237 4 Place Jussieu F-75252 Paris Cedex 05France

Sorbonne Universites UPMC Univ Paris 06 Faculte de BiologieUnite de Recherche UPMC UR6 lsquolsquoEnzymologie de lrsquoARNrsquorsquo 4 PlaceJussieu F-75252 Paris Cedex 05 France Tel +33-1-44-27-40-86Fax +33-1-44-27-61-51 email codjohountondjiupmcfr

In this report we have used periodate-oxidized tRNA(tRNAox) as an affinity laleling reagent to demonstratethat (i) the bL12 protein contacts the CCA-arm of P-site bound tRNA on the Escherichia coli 70S ribo-somes (ii) the stoichiometry of labelling is one moleculeof tRNAox bound to one polypeptide chain of endogen-ous bL12 (iii) cross-linking in situ of bL12 withtRNAox on the ribosomes provokes the loss of activity(iv) intact tRNA protects bL12 in the 70S ribosomesagainst cross-linking with tRNAox (v) both tRNAoxand pyridoxal 50-phosphate (PLP) compete for thesame or for proximal cross-linking site(s) on bL12inside the ribosome (vi) the stoichiometry of cross-link-ing of PLP to the recombinant E coli bL12 protein isone molecule of PLP covalently bound per polypeptidechain (vii) the amino acid residue of recombinant bL12cross-linked with PLP is Lys-65 (viii) Lys-65 of E colibL12 corresponds to Lys-53 of eL42 which was previ-ously shown to cross-link with P-site bound tRNAox onhuman 80S ribosomes in situ (ix) finally E coli bL12and human eL42 proteins display significant primarystructure similarities which argues for evolutionaryconservation of these two proteins located at thetRNA-CCA binding site on eubacterial and eukaryalribosomes

Keywords E coli 70S ribosomes E coli ribosomalprotein bL12 Lys-65 of bL12 periodate-oxidizedtRNA tRNA-CCA binding site

Abbreviations bL12 (formerly L12) eubacterial largesubunit ribosomal protein L12 with a free NH2 ter-minus according to the new system for namingribosomal proteins L7 the NH2-terminally acety-lated bL12 eL42 eukaryal or archaeal large subunitribosomal protein L42 (formerly L42A or L42AB inyeast or L36a in human or L44e in archaea) PLPpyridoxal 5rsquo-phosphate PMP pyridoxamine 5rsquo-phos-phate PTC peptidyl transferase centre rP ribosomal

protein tRNAox periodate-oxidized tRNA the 2030-dialdehyde derivative of tRNA uL10 the largesubunit ribosomal protein L10 present in the threemajor kingdoms of life

In all kingdoms of life the ribosome consists of a largeand a small subunits each of which having separatefunctions Peptide bond formation occurs at the pepti-dyl transferase centre (PTC) of the large subunit (50Sin eubacteria and archaeabacteria and 60S in eukary-otes) whereas mRNA sequences are decoded on thesmall subunit (30S in bacteria and 40S in eukaryotes)

The current view of the PTC is that it is composedsolely of RNA and that no protein is involved in themechanism of peptide bond formation (1 2) This viewstemmed from the observation of the 3D structure ofthe 50S ribosomal subunit of Haloarcula marismortuiwhich reveals a void of protein electron density in aradius of 18 angstroms of the PTC (1 3) Howevercrystallographic data on the full 70S ribosome fromThermus thermophilus show that ribosomal proteinL27 extends with its N-terminus into the PTC andmakes contact with the tRNA substrates (4) In ac-cordance with these structural data Polikanov et al(5) have recently proposed that the N-terminus of Tthermophilus L27 might take part in peptide bond for-mation In sharp contrast with these structural datait was recently demonstrated that L27 doesnrsquot playany significant role in peptide-bond formation (6)Moreover ribosomes from archaea or eukaryotes donot have protein L27 or any homologous counterpartindicating that L27 cannot be a part of an evolution-arily conserved peptidyl transfer mechanism

It should be recalled that in the past all steps of thetranslation process including the peptidyl transferstep were thought to be supported by proteins(712) Accordingly early biochemical studies haddemonstrated that peptidyl transferase is an enzymewith the following characteristics (i) the reaction ofpeptidyl transfer has a pH dependence with a pKa of72 which was supposed to reflect the presence onthe enzyme of an ionizable functional group such ashistidine (711) (ii) ribosomal inactivation by ethox-yformic anhydride (a reagent specific for His) was op-timal at pH 70 (8) while photochemical inactivationof the ribosome with Rose Bengal dye exhibited a pHoptimal at 75 (9) (iii) other small-molecule inhibitorsof the activity of the ribosome known to target aminoacid side chains include pyridoxal 5rsquo-phosphate (PLP)which inactivates Escherichia coli ribosomes by

J Biochem 2017112 doi101093jbmvx055

The Authors 2017 Published by Oxford University Press on behalf of the Japanese Biochemical Society All rights reserved 1Downloaded from httpsacademicoupcomjbarticle-abstractdoi101093jbmvx0554082913Affinity-labelling-in-situ-of-the-bL12-protein-onby BIUSJ (Paris 6) useron 17 October 2017

covalently binding in majority to the L7L12 protein(12) However the amino acid residue(s) targeted bythese reagents on the ribosome had not been identifiedSince to date structural and biochemical studies failedto identify any ribosomal group that acts in chemicalcatalysis of peptide bond formation (112) new toolsare needed Among these affinity labelling represents asuitable strategy for the identification of the aminoacid residues involved in the molecular mechanismsof biological processes In this context we have previ-ously demonstrated that the lysyl residue 53 of thehuman large subunit ribosomal protein eL42 couldbe affinity labelled with periodate-oxidized tRNA(tRNAox) the 2030-dialdehyde derivative of tRNA atthe PE hybrid site and that this covalent reactioncompletely abolished the poly(Phe) synthesis activityof the human 80S ribosomes (13) This result suggeststhat Lys-53 might play a functional role Since the eu-bacterial ribosomes do not have the eukaryote-specificeL42 protein or any homologous counterpart we haveapplied in the present report the same affinity labellingstrategy to E coli ribosomes with the goal of identify-ing the protein(s) located at the tRNA-CCA bindingsite on these eubacterial ribosomes Here we demon-strate that the E coli large subunit ribosomal proteinbL12 can be affinity labeled with tRNAox and thatthis 2030-dialdehyde derivative of tRNA fulfills all thecriteria for a site-directed affinity label of the bL12protein inside the E coli 70S ribosomes Notably (i)the stoichiometry of covalent binding of tRNAox tobL12 is one molecule of tRNAox bound to one poly-peptide chain of endogenous bL12 (ii) cross-linking insitu of bL12 with tRNAox on the E coli ribosomesprovokes the loss of activity (iii) intact tRNA protectsbL12 in the E coli 70S ribosomes against cross-linkingin situ with tRNAox (iv) both tRNAox and PLP com-pete for the same or for proximal cross-linking site(s)on bL12 inside the ribosome while the amino acidresidue of recombinant bL12 cross-linked with PLPis Lys-65 (v) Lys-65 of the 62GANK65 motif of Ecoli bL12 corresponds to Lys-53 of the 49GGQTK53motif of the large subunit ribosomal protein eL42which was previously shown to cross-link with P-sitebound tRNAox on human 80S ribosomes in situ Notethat the new system for naming ribosomal proteins(14) is used in this report Accordingly the eubacteriallarge subunit ribosomal protein L12 with a free NH2terminus as well as the NH2-terminally acetylated L7chain whose amino acid sequence is identical to that ofL12 are both called bL12 Similarly the eukaryal orarchaeal large subunit ribosomal proteins of the L44Efamily (formerly L42A or L42AB in yeast or L36a inhuman or L44e in archaea) are called eL42 (14)

Materials and Methods

MaterialsE coli 70S ribosomes were obtained as already described (15) 40Sand 60S ribosomal subunits with intact rRNAs were isolated fromunfrozen human placenta (16) 80S ribosomes were obtained by as-sociation of re-activated 40S and 60S subunits and used in controlcross-linking experiments as previously reported in (13 17)Recombinant large subunit ribosomal protein bL12 from E coliwas purified from the overproducing strain BL21(DE3) carrying

recombinant plasmid pET21L12C-6His (a generous gift of Dr SSanyal) The purification procedure consisted mainly in a chroma-tographic step on a Ni-NTA superflow column (Qiagen) PLP waspurchased from Sigma Aldrich Trypsin was from Promega Theshort mRNA analogues (oligoribonucleotides GAA UUU GACAAA and GAA AUG GAC AAA) as well as E coli tRNAPhe

were purchased from Sigma Aldrich tRNAAsp from yeast was pur-ified by counter-current chromatography followed by separation bypolyacrylamide gel electrophoresis Its amino acid acceptance cap-acity was 1400 pmolA260 unit

32P-labelling of tRNA at the 50-endwas carried out after dephosphorylation by alkaline phosphatase(Roche) in the presence of g-[32 P]ATP and polynucleotide kinasetRNAox was prepared as described previously in (13 17)

Methods

Cross-linking of E coli 70S ribosomes with tRNAox or PLP Onone hand E coli 70S ribosomes (05 mM) were incubated at 37 C inbuffer A (50mM TrisHCl pH 75 100mM KCl 10mM MgCl2and 05mM EDTA) containing 5mM of the dodecaribonucleotideGAA UUUGAC AAA as an mRNA in a total volume of 25 ml Thelabelling reaction was initiated by the addition of 10 mM[32P]tRNAAspox and 5mM sodium cyanoborohydride(NaBH3CN) and the incubation continued for 1 h After the termin-ation of the tRNAox-labelling reaction the incubation mixtureswere quenched by the addition of 1 ml of 05 M sodium borohydride(NaBH4) freshly prepared in 50mM phosphate buffer (pH 75) 20 mlportions of the NaBH4-quenched samples were applied onto a 10polyacrylamide gel which was run by SDS-gel electrophoresis Thegels were dried under vacuum and subjected to autoradiographyControl experiments for cross-linking of ribosomal proteins withtRNAox contained [32 P]tRNAAspox (10 mM) incubated in thesame conditions in the absence of ribosomes tRNAox-labelling ofrecombinant E coli bL12 (2mM) with [32P]tRNAAspox (10 mM) wasperformed in the same conditions as for the ribosomes In order toidentify the ribosomal proteins cross-linked in situ with tRNAox bymass spectrometric analyses the 10 polyacrylamide gel was run byUrea-gel electrophoresis for the preparation of those proteins Onthe other hand E coli 70S ribosomes (05 mM) in 25 ml were pre-incubated with 1mM PLP 1mM pyridoxamine 50-phosphate (PMP)or 20 mM native tRNAAsp at 25 C in buffer A prior to the additionof [32P]tRNAAspox (10 mM)

Poly(Phe) synthesis activity of E coli 70 S ribosomes Poly(Phe)synthesis was determined as incorporation of L-[3H]Phenylalanineinto hot trichloroacetic acid-insoluble material as previouslydescribed in (13) The final reaction mixture (70 ml) contained40mM Tris-HCl (pH 75) 7mM MgCl2 80mM NH4Cl 1mMdithiothreitol 1mM ATP 1mM phosphoenolpyruvate 05mMGTP 50 mgml pyruvate kinase 25 mM tRNAPhe (first chargedduring a 30min incubation at 30 C with a 3 fold excess ofL-[3 H]Phenylalanine (37GBqmmol) and a saturating amountof partially purified phenylalanyl-tRNA synthetase) 6 mg poly(U)05 mM EF-Tu 02 mM EF-Ts and 03 mM EF-G To check the effectof the cross-linking of the E coli 70S or the human 80S ribosomeswith tRNAPheox on the poly(U)-dependent synthesis of poly(Phe)the reaction (70 ml) was started with 14 ml of mixture containing14 pmol of the E coli 70S or the human 80S ribosomes that werecross-linked with tRNAPheox with final concentrations of 03 mMribosomes and 44 mM tRNAs The concentrations of elongationfactors with the eukaryotic 80S dependent poly(Phe) synthesiswere 05 mM EF-1a 015 mM EF-1b and 035 mM EF-2 Duringincubation at 30 C with the E coli system or at 37 C with theeukaryotic system 15 ml samples were withdrawn at indicatedtimes and spotted on glass fibre filters and hot trichloroacetic acidinsoluble radioactivity was determined

Cross-linking of recombinant E coli bL12 protein with PLPRecombinant bL12 protein (350 mM) in 100 ml was incubated withPLP (350 mM) during 30min at 30 C in 50mM phosphate buffer(pH 75) The incubation mixtures were quenched by the addition of1 ml of 2 M sodium borohydride (NaBH4) freshly prepared in 50mMphosphate buffer (pH 75) The NaBH4-quenched samples (100 ml)were applied to a Superdex 75 column 10 300 GL (GampEHealthcare) equilibrated in 50mM sodium phosphate buffer (pH75) containing 150mM NaCl The flow rate was 04mlmin

C Hountondji et al

2Downloaded from httpsacademicoupcomjbarticle-abstractdoi101093jbmvx0554082913Affinity-labelling-in-situ-of-the-bL12-protein-onby BIUSJ (Paris 6) useron 17 October 2017

Elution was monitored by the absorbance at 280 nm (for the protein)and 325 nm (for the covalently bound PLP) (18 19)

Sample preparation for mass spectrometric analyses After elutionfrom the Superdex column the phosphopyridoxylated recombinantbL12 protein absorbing at 325 nm was analysed by ESI mass spec-trometry in order to measure the stoichiometry of cross-linking withPLP or digested with trypsin prior to mass spectrometric analysis tosearch for the phosphopyridoxylated peptides of bL12

Analysis by ESI mass spectrometry of bL12 cross-linked with PLPSolutions of bL12 protein or of bL12-PLP covalent complex weredesalted using a Ziptip C4 (Millipore Saint-Quentin-en-YvelinesFrance) the proteins were eluted with 10 ml of a solution of 39water 1 formic acid and 60 acetonitrile These eluates wereloaded into NanoESI spray capillaries (Thermo Fisher ScientificLes Ulis France) A 15 kV voltage was applied on the capillary toproduce a stable spray in front of an orbitrap mass spectrometerAnalyses of the multi-charged ions produced were done in the orbi-trap (Thermo Fisher Scientific) settled with a resolution of 100000 atmz 400 Multi-charged spectra were deconvoluted using the Xtractsoftware (Thermo Fisher Scientific) in order to determine the mo-lecular weight of the protein and the covalent complex

Analysis of the tryptic digest of the bL12-PLP complex by MALDIMS The covalent complex was digested overnight by trypsin in a01 M ammonium carbonate buffer After digestion the solution wasdesalted using a Ziptip C18 and the peptides were eluted and loadedon a MALDI plate using a solution of 5mgml of a-Cyano-4-hydro-xycinnamic acid in 30 (water 01 TFA) 70 acetonitrileMALDI-TOF MS and MALDI-TOFTOF MSMS analyses wereperformed using a 5800 MALDI-TOFTOF mass spectrometer (ABSciex Les Ulis France) The instrument was equipped with anNdYAG laser (operating at 355 nm wavelength 500 ps pulse and200Hz repetition rate) Acquisitions were performed in the positiveion mode MS were processed using DataExplorer 44 (AB Sciex)

Results

Affinity labelling in situ of rp bL12 with tRNAox on theE coli 70S ribosomesWe had previously used tRNAox as an affinity label-ling reagent to identify lysine and arginine residues at

the tRNA-CCA binding site on enzymes of the trans-lation apparatus especially the aminoacyl-tRNAsynthetases (2025) This 2030-dialdehyde derivativeof tRNA had been obtained by the periodate treatmentof native tRNA which specifically oxidizes the 2030-cis-diol function of the 30-terminal ribose of tRNAThis approach had led to the discovery of theKMSKS motif that is considered today as a signaturefor the active site of class 1 aminoacyl-tRNA synthe-tases (26) More recently we have demonstrated thatthe lysyl residue 53 of the human large subunit riboso-mal protein eL42 could be affinity labelled withtRNAox at the PE hybrid site and that this covalentreaction completely abolished the poly(Phe) synthesisactivity of the human 80S ribosomes suggesting thatLys-53 might play a functional role (13) We haveapplied in the present report the same affinity labellingstrategy to E coli ribosomes with the goal of identify-ing lysine or arginine residues at the binding site for theCCA arm of tRNA

When we incubated E coli 70S ribosomes or theHuman 80S ribosomes (05 mM) in the presence ofthe dodecaribonucleotide GAAUUUGACAAA asmRNA (5 mM) and of 10 mM [32P]tRNAAspox pos-itioned at the P-site only one ribosomal protein wasfound cross-linked in E coli and in human respect-ively as revealed by the analysis of the incubation mix-tures of cross-linking by SDS-PAGE on 10polyacrylamide gels (Fig 1A and (13 17)) The radio-active band corresponding to unreacted [32P]tRNAoxin the reaction mixtures was assigned by the position ofa control [32P]tRNAAspox loaded alone in Lane 3 (Fig1A) These proteins were identified as the E coli largesubunit ribosomal protein bL12 and the human eL42protein respectively by MALDI mass spectrometricanalyses performed as described in (27) In the caseof the E coli bL12 protein five peptides corresponding

Fig 1 Autoradiogram of the SDS gel electrophoresis analysis in 10 polyacrylamide of [32P]tRNAAspox-labelled endogenous proteins of E coli70S or human 80S ribosomes The tRNAox-labelling incubation mixtures were as described in Materials and Methods (A) Lane 1 E coli 70Sribosomes incubated with [32P]tRNAAspox The protein contained in the rP-tRNAox band in Lane 1 was identified as the large subunitribosomal protein bL12 by mass spectrometry NanoLC-MSMS (see text) Lane 2 human 80S ribosomes incubated with [32P]tRNAAspox Theprotein contained in the rP-tRNAox band in Lane 2 was identified as the human large subunit ribosomal protein eL42 (see text) Lane 3[32P]tRNAAspox alone (B) Lane 1 E coli 70S ribosomes (05 mM) incubated with [32P]tRNAAspox (10 mM) Lane 2 E coli 70S ribosomes(05 mM) incubated with 1mM PMP prior to the addition of [32P]tRNAAspox (10mM) Lane 3 E coli 70S ribosomes (05 mM) incubated with amixture of PLP (02mM) and [32P]tRNAAspox (10 mM) Lane 4 E coli 70S ribosomes incubated with PLP (1mM) during 30min prior to theaddition of [32P]tRNAAspox (10mM) Lane 5 E coli 70S ribosomes incubated with [32P]tRNAAspox-red (10 mM) the [32P]tRNAAsp-dialdehydederivative reduced with NaBH4 Lane 6 protection with native tRNAAsp (20 mM) against labelling by [32P]tRNAAspox of ribosomal protein bL12inside the E coli 70S ribosomes Incubation time was 1 h

The E coli rP bL12 contacts the CCA-end of P-site tRNA

3Downloaded from httpsacademicoupcomjbarticle-abstractdoi101093jbmvx0554082913Affinity-labelling-in-situ-of-the-bL12-protein-onby BIUSJ (Paris 6) useron 17 October 2017

to about 50 of the sequence coverage were foundafter in-gel digestion of the Urea-PAGE rP-tRNAoxgel band in Lane 1 of Figure 1A followed by analysisby MALDI mass spectrometry The amino acid se-quences deduced from the analyses are shown inSupplementary Table S1 As for eL42 (used here as acontrol in Lane 2 of Fig 1A) the identification thatwas already reported (13 17) was reproduciblyachieved in this study (results not shown) As previ-ously described for the preparation of ribosomal pro-teins cross-linked in situ with tRNAox in order toidentify those proteins by mass spectrometric analysesthe 10 polyacrylamide gel was run by urea-gel elec-trophoresis Indeed as discussed in a previous report(17) an advantage in running polyacrylamide gel elec-trophoresis in urea is that the majority of the rPs mi-grate towards the () pole because they are positivelycharged whereas rP-tRNAox covalent complexes withtheir additional 75 negative charges of the phosphategroups of tRNA migrate in the opposite direction Asa consequence the rP-[32P]tRNAox band can be easilydetected alone on the Urea-PAGE gel

tRNAox is a site-directed affinity label of bL12 in the Ecoli 70S ribosomesOur data show that tRNAox fulfills all the criteria fora site-directed affinity label of the bL12 protein insidethe E coli 70S ribosomes As shown in Figure 1 thebL12-tRNAox covalent complex has an apparent mo-lecular weight of 36000 plusmn 1000Da that agreed wellwith the one expected for a covalent complex contain-ing one polypeptide chain of endogenous E coli bL12(about 12000Da) and one molecule of tRNAox(25000Da calculated MW of the binary rP-tRNAoxcomplex 37000Da) Interestingly the eL42-tRNAoxcovalent complex has also an apparent molecularweight of 36000 plusmn 1000Da comprising one moleculeof eL42 (molecular weight 12000Da) and one mol-ecule of tRNAox (25000Da) as previously reported(Fig 1 and (13 17)) The 11 stoichiometry of covalentbinding of tRNAox to bL12 is compatible with thepresence of one binding site for the CCA-arm oftRNA and for the translation factors in the C-ter-minal domain (CTD) of each of the four subunits ofthe protein In fact the bL12 protein from the E coli70S ribosomes is composed of two dimers bound toone copy of protein uL10 (formerly L10) which an-chors the pentamer to the large 50S ribosomal subunit(2830) Each monomer of bL12 contains one bindingsite for tRNA and for the elongation factors (31) Inaddition intact tRNA completely protected bL12 inthe E coli 70S ribosomes against cross-linking in situwith tRNAox (Lane 6 in Fig 1B) Since it is wellknown that native tRNA binds first to the P-site thisresult might reflect competition between nativetRNAAsp and the dialdehyde derivative thereof forthe binding to the P-site This result suggests at thesame time that the tRNA-bL12 covalent complex for-mation occurred specifically at the P-site of 70S ribo-somes It should be recalled that the 2030-dialdehydederivative of tRNA had also been previously shown tobind to the P-site as follows [32 P]tRNAfMetox from Ecoli was positioned onto the P-site of E coli 70S

ribosomes in the presence of the mRNA analogue GAAAUGGACAAA containing the universal P-sitetriplet AUG and the same band representing thebL12-tRNAox covalent complex was observed on thepolyacrylamide gel (32) This result strongly suggeststhat the 2rsquo3rsquo-dialdehyde derivative of [32P]tRNAfMet isbound to the P-site of E coli 70S ribosomes in a tern-ary complex with the AUG codon of mRNA (32)However at that time the protein labelled bytRNAox on the E coli 70S ribosomes could not beidentified due to technical problems (32) Finallycross-linking in situ of bL12 with tRNAox in the Ecoli 70S ribosomes provokes the loss of the activity ofpoly(U)-dependent poly(Phe) synthesis suggestingthat the lysyl residue cross-linked with tRNAoxmight play a functional role in eubacterial ribosomes(Fig 2)

Affinity labelling in situ of rP bL12 with tRNAox on theE coli 70S ribosomes with or without PLPAs previously reported in the case of human eL42MALDI mass spectrometric identification of

A

B

Fig 2 Effect of tRNAox-labelling of the E coli bL12 or the human

eL42 proteins at the P-site on poly(Phe) synthesis by poly(U)-pro-

grammed E coli or human ribosomes Values on the ordinate axisrepresent the amount (pmol) of [3H]Phenylalanine incorporated intothe poly(Phe) polypeptide in the presence of tRNAPheox (triangles)or of native tRNAPhe (circles) For details see Materials andMethodsrsquo Section

C Hountondji et al

4Downloaded from httpsacademicoupcomjbarticle-abstractdoi101093jbmvx0554082913Affinity-labelling-in-situ-of-the-bL12-protein-onby BIUSJ (Paris 6) useron 17 October 2017

tRNAox-labelled peptides turned out to be verydifficult due to the weakness of the signal of thecross-linked peptide and to mass deviations betweencalculated and measured masses Therefore theamino acid side chains targeted by tRNAox were iden-tified indirectly by allowing this tRNA analogue tocompete through the cross-linking reaction withsmall-molecule inhibitors of the activity of the ribo-some whose cross-linking sites on bL12 can be moreeasily identified Among these molecules PLP hadbeen previously shown to inactivate E coli ribosomesby covalently binding in majority to the bL12 protein(12) Interestingly it was previously shown that bothtRNAox and PLP proceed by Schiff base formationwith the side chain of a lysine (12 18 20 27) suggest-ing that they might target the same amino acid residueson bL12 Competition between tRNAox and PLP forthe cross-linking with bL12 is supported by the dem-onstration that in the affinity labelling reaction ofaminoacyl-tRNA synthetases with tRNAox in the1980s there was generally only one lysine residuelabelled in majority at the enzyme active site that ex-hibited a higher reactivity through Schiff base forma-tion due to an abnormally low pKa value (21 24 25)This reactive lysine residue was always found equallycross-linked with tRNAox or with PLP (18 21 24 25)As shown in Figure 1B (Lane 5) treatment of[32P]tRNAAspox with NaBH4 yielded a reduced[32P]tRNAAspox-red derivative which failed to cross-link with bL12 on the E coli 70S ribosomes This resultsuggests that cross-linking of this protein withtRNAox proceeds through the formation of a Schiffbase as expected Moreover preincubation of E coli70S ribosomes with 1mM PLP during 30min prior tothe addition of [32P]tRNAAspox prevented the cross-linking reaction in situ between bL12 and the tRNAdialdehyde derivative (Lane 4 in Fig 1B) This result iscompatible with the cross-linking of PLP proceedingequally through the formation of a Schiff base as ex-pected Interestingly as previously proposed byOhsawa et al (12) the cross-linking reaction in situbetween PLP and the bL12 protein on the E coli 70Sribosomes was shown to proceed through the forma-tion of a Schiff base by the following observationPMP which lacks the aldehyde group capable of form-ing a Schiff base with a lysyl e-amino group could notprevent the cross-linking reaction in situ between bL12and tRNAox (Lane 2 in Fig 1B) Altogether theabove-cited data suggest that both PLP and tRNAoxare capable of cross-linking with the e-amino group ofthe same lysyl residue(s) of bL12 Competition be-tween PLP and tRNAox for the cross-linking ofbL12 was further demonstrated by the fact that inthe presence of 02mM PLP and 5 mM[32P]tRNAAspox the cross-linking yield is decreased(Lane 3 in Fig 1B) We have used this PLP concentra-tion because it corresponds to the KD(app) that is theconcentration for half-maximal amount of PLP-labelled lysyl residues at the phosphate binding siteon the aminoacyl-tRNA synthetases (18) Applicationof such a KD(app) value to the in situ cross-linking ofbL12 with PLP on E coli 70S ribosomes would explain

why the yield of cross-linking with tRNAox wasdecreased (Lane 3 in Fig 1B)

Analysis by mass spectrometry of bL12 cross-linkedwith PLPThe possibility that tRNAox and PLP might competefor the same cross-linking site(s) on the E coli 70Sribosomes prompted us to search for the lysyl resi-due(s) cross-linked with PLP on bL12 However a pre-requisite for this experiment is to verify that bL12effectively cross-links with PLP as previously reportedby the group of Gualerzi (12) For this purpose wehave used the recombinant protein rather than thewhole ribosome for the following reasons (i) the tern-ary and quaternary structures of the bL12 protein asdeduced from intensive structural studies in solutionand in the ribosome or from the crystal structure ofthe whole protein were previously shown to be com-parable in solution and in the ribosome suggestingthat the 3D structure of the pentameric (bL12)4-uL10complex is independent of the whole 50S ribosomalsubunit (2931 33 34) (ii) protein bL12 can beremoved easily and selectively from the large riboso-mal subunit (35) (iii) wild-type bL12 can be replacedby variant bL12 proteins (35) Taking advantage ofthis special feature Sanyalrsquos group in Uppsala(Sweden) has constructed a novel E coli strain JE28 in which a (His)6-tag has been inserted at the C-terminus of bL12 (35) These data suggest that thebL12 protein might behave similarly on and off theribosome and that the extent of PLP incorporationinto this protein would be most probably the sameon and off the E coli 70S ribosomes The calculatedmolecular mass of the polypeptide chain of the recom-binant E coli bL12 protein used in this study was ob-tained as follows (i) the molecular mass of the E colibL12 polypeptide chain without any post-translationalmodification was 12295Da as indicated in the ProteinData Bank (ii) this polypeptide chain was His-tagged(at the C-terminus) with the sequence Leu-Glu-(His)6of molecular mass 1064Da giving rise to a His-taggedbL12 protein of molecular mass 13359Da (iii) sincethis recombinant protein was expressed in an E colistrain post-translational modification by removal ofthe N-terminal methionine (131Da) would result ina mature bL12 polypeptide chain of molecularmass 13228Da Supplementary Figure S1 showsthe ESI orbitrap spectrum of the bL12 protein(Supplementary Fig S1A) as well as the one of thebL12-PLP covalent complex (Supplementary FigS1B) The monoisotopic mass of the bL12 proteinwas found equal to the calculated monositopic mass1322095Da (Supplementary Fig S2) correspondingto the E coli bL12 polypeptide chain with a free NH2

terminus His-tagged (at the C-terminus) with the se-quence Leu-Glu-(His)6 and lacking the N-terminal me-thionine Determination of the molar ratio of boundPLP to bL12 as well as the identification of peptidesmodified by PLP were achieved simply by measuringthe mass increase due to PLP (molecular mass of231Da) Supplementary Figure S2 shows the massspectra of the control recombinant bL12 protein(Supplementary Fig S2A) and of the bL12 protein

The E coli rP bL12 contacts the CCA-end of P-site tRNA

5Downloaded from httpsacademicoupcomjbarticle-abstractdoi101093jbmvx0554082913Affinity-labelling-in-situ-of-the-bL12-protein-onby BIUSJ (Paris 6) useron 17 October 2017

cross-linked with PLP (Supplementary Fig S2B) Onone hand the peak of monoisotopic mass 1345196Dashowed a mass value increased by 231Da relative tothe 1322094Da peak corresponding to the molecularmass of the monomer bL12 with a free NH2 terminusindicating that the bL12 polypeptide chain is cross-linked with one molecule of PLP (SupplementaryFig S2B) On the other hand the 1349397Da andthe 1326295Da peaks showed each a mass valueincreased by 4142Da relative to the 1345196Da (co-valent bL12-PLP complex) and the 1322094Da(bL12) peaks indicating that the NH2-terminallyacetylated polypeptide chain is also crosslinked withone molecule of PLP (Supplementary Fig S2B) Inconclusion each polypeptide chain of bL12 wasfound to be cross-linked with one PLP moleculeHowever the presence of both acetylated and nonacetylated polypeptides that were not modified byPLP (Supplementary Fig S2B) would suggest thatnot all the four chains of the tetramer are cross-linked with PLP This situation is likely to be the con-sequence of the chemical modification of the recombin-ant bL12 protein (350 mM) with non-saturatingamounts of PLP (350 mM) The same result was ob-tained when bL12 (350mM) was allowed to reactwith 1mM PLP

Identification of the amino acid side chains targetedby PLP on the recombinant E coli bL12 proteinMass spectrometric analysis of tryptic peptides from350 mM bL12 cross-linked with 350 mM PLP gave a[M + H]+ signal at mz 127267 presenting a massvalue increased by 231Da (the molecular mass of PLP)relative to unmodified peptide with a calculated mass104164Da corresponding to stretch 60AAGANKV

AVIK70 (results not shown) Non cleavage of the pep-tide bond between Lys-65 and Val-66 implies that Lys-65 is actually the amino acid residue whose side chainis covalently labelled with PLP Another peptide cross-linked with PLP was found at mz 159888 (Fig 3) Itmatched with fragment 60AAGANKVAVIKAVR73(theoretical mass of 136785Da) whose mass wasincreased by 231Da (Fig 3) Owing to the 11 stoichi-ometry of modification of the protein with PLP onLys-65 (Supplementary Fig S2B) this fragment wasassumed to result from a miscleavage at Lys-70Finally the 150291Da peak (Fig 3) resulted from ametastable fragment characteristic of the loss of thephosphate group (98Da) of PLP (36) from the159888Da fragment and may be considered as a sig-nature of peptides cross-linked with PLP It should benoted that when bL12 (50 mM) was cross-linked with1mM PLP only Lys-65 was found cross-linked withthe reagent

pK of the tRNAox-labelled lys-65 residueWe had previously demonstrated that the ribosomalprotein eL42 could be affinity labelled in situ withtRNAox on the human 80S ribosomes (13 17 27)We had also demonstrated that the human recombin-ant eL42 protein could be equally affinity labelled withtRNAox (27) We had concluded that human eL42 isthe only ribosomal protein that can interact withtRNA on and off the ribosome (27) Affinity labellingin situ with tRNAox of the eL42 protein from thehuman 80S ribosomes as a function of the pH of theincubation mixture indicated a pK value of 69 plusmn 02for Lys-53 (27) Similarly affinity labelling withtRNAox of the human recombinant eL42 protein asa function of the pH indicated a pK value of 69 plusmn 01

Fig 3 MALDI mass fingerprint of tryptic peptides from bL12 cross-linked with PLP The sequence coverage by the trypsin was 97 of thetheoretical sequence The main peaks are annotated The stars correspond respectively to peptides 66VAVIKAVR73 (theoretical mass of85558Da) and 60AAGANKVAVIKAVR73 (theoretical mass of 136785Da) whose mass was increased by 231Da The zoom on the peak15987Da shows the metastable fragmentation of the peptide cross-linked with PLP

C Hountondji et al

6Downloaded from httpsacademicoupcomjbarticle-abstractdoi101093jbmvx0554082913Affinity-labelling-in-situ-of-the-bL12-protein-onby BIUSJ (Paris 6) useron 17 October 2017

for Lys-53 (unpublished data) In the present reportwe have applied these affinity labelling approaches tothe bL12 protein on and off the E coli 70S ribosomesin order to check whether this eubacterial protein hasthe same behaviour as that of the eukaryal eL42 Wehave followed the tRNAox-labelling reaction betweenthe free E coli recombinant bL12 protein and[32P]tRNAox as a function of the pH Figure 4Ashows the incorporation of [32P]tRNAox into the re-combinant bL12 as a function of the pH of the incu-bation mixture The data from Figure 4A were used toplot the molar fraction of [32P]tRNAox incorporatedinto (or cross-linked with) recombinant E coli bL12versus increasing pH values and a pK value of 72 plusmn02 was deduced for the e-amino group of Lys-65 (Fig4B) [32P]tRNAox incorporation into bL12 inside theE coli 70S ribosomes as a function of the pH could bealso followed It indicated a pK value of 72 plusmn 01 forLys-65 (results not shown) Similar pK values for thee-amino group of Lys-65 found with the free recom-binant and the ribosome-bound bL12 protein suggestthat similarly to human eL42 (27) bL12 is capable ofinteracting with tRNA on and off the ribosome ThepK value of 72 which is 3 units lower than is normallyfound for the side chain of a lysine residue (pK 105)describes the dissociation of the protonated form ofthe e-amino group of Lys-65 and its greater reactivitythrough the tRNAox-labelling reaction in accordancewith the data obtained with eL42 from human 80Sribosomes

Common features of the E coli bL12 and the humaneL42 proteinsFirst these two proteins have comparable apparentmolecular weights of about 12000Da Comparisonof the migration of the cross-linked complexes fromhuman or E coli ribosomes to that of protein markerson the same gels indicated apparent molecular weightsof 36000 plusmn 1000Da (Fig 1) These molecular weightsagreed well with that expected for a covalent complex

containing one molecule of endogenous human eL42or E coli bL12 (about 12000Da each according tothe PDB) and one molecule of tRNAox (25000Dacalculated MW of the binary rP-tRNAox complex37000Da) Second as shown in Figure 2 cross-link-ing in situ of these proteins with tRNAox in the E coli70S or the human 80S ribosomes provokes the loss ofthe activity of poly(U)-dependent poly(Phe) synthesissuggesting that the lysyl residues cross-linked withtRNAox might play a functional role in eubacterialor eukaryal ribosomes respectively Since bL12 is theonly E coli large subunit ribosomal protein cross-linked with tRNAox similarly to the human eL42protein one can conclude that cross-linking of theseproteins with tRNAox is directly or indirectly respon-sible for the activity loss observed in Figure 2 as al-ready proposed for human eL42 in a previous report(12) This conclusion corroborates the finding that theremoval of bL12 from E coli 70S ribosomes reducesthe rate of protein synthesis (35) However it has beenshown in a previous study that the mutation K65A inbL12 reduces the binding affinity to the ternary com-plex EF-TuGTPaminoacyl-tRNA by only a factor of4 which is a very moderate effect (37) Third theydisplay significant primary structure similarities withan average of 40-50 of identities and conservativereplacements depending on the insertion of gaps intothe amino acid sequences (Fig 5A) In particular the62GANK65 motif of bL12 the lysyl residue of whichis shown to cross-link with PLP in the present reportmatches with the 49GGQTK53 motif of eL42 (Fig5B) The latter motif was found cross-linked withtRNAox through the formation of a Schiff base withLys-53 in a previous study (12) The score of sequencesimilarity around the GANK motif was about 63(Fig 5B) Proteins eL42 are only found in archaeaand eukaryotes As previously reported alignment ofthe amino acid sequences of eukaryotic eL42 proteinswith those of the archaebacterial counterparts indi-cates only 44 average amino acid identities and

A B

Fig 4 Labelling of recombinant E coli bL12 with [32 P]tRNAox as a function of the pH of the incubation mixture (A) the incubation mixtureswere analysed by SDS-PAGE on 10 polyacrylamide gels and the yields of labelling were calculated as described earlier in (27) The tRNAox-bL12 covalent complex formed is shown Lanes 1-6 correspond to pH 47 58 65 74 83 and 94 respectively (B) plot of the molar fraction of[32P]tRNAox incorporated into (or crosslinked with) recombinant E coli bL12 as a function of the pH of the incubation mixture The extents ofincorporation of [32P]tRNAox at pH 47 58 65 74 83 and 94 were determined from (A) Two datasets from two separate experiments as in(A) were used to obtain this graph The data were fitted with Mathematica to the function inscribed in the figure with the following features thedashed lines defines the 95 CIs for the prediction of a single value whereas the continous lines and the gray-shaded region define the 95 CIsfor the prediction of the average curve with pK=72 plusmn 02

The E coli rP bL12 contacts the CCA-end of P-site tRNA

7Downloaded from httpsacademicoupcomjbarticle-abstractdoi101093jbmvx0554082913Affinity-labelling-in-situ-of-the-bL12-protein-onby BIUSJ (Paris 6) useron 17 October 2017

conservative replacements (17) Since it is generallybelieved that proteins sharing more than 30 primarystructure similarity may be phylogenetically relatedthe 4050 of identities and conservative replace-ments between eL42 and bL12 reported here (Fig5A) might reflect an evolutionary conservation of theeubacterial bL12 and the eukaryotic or the archaealeL42 ribosomal proteins Fourth the tRNAox-labelledLys-65 of E coli bL12 and Lys-53 of human eL42 ex-hibit an abnormally low pK as compared with the onethat is normally found for the side chain of a lysineresidue (pK 105) These results are consistent with thefact that both residues are reactive toward the reactionwith tRNAox Fifth the GANK and GGQTK motifsoccupy respectively comparable positions in the 3Dstructures of these proteins (Fig 6A and B) In the3D structure of human eL42 modelled by homologywith the archaeal counterpart fromH marismortui theGGQTK peptide is located in an extended loop at oneextremity ((27) and Fig 6B) Similarly the GANKpeptide is located at the tip of the crystallographicstructure of the CTD of bL12 ((30 31 33) and Fig6A) Altogether these structural similarities argue for

the evolutionary conservation of the eL42 and bL12proteins located at the tRNA-CCA binding site oneukaryal or eubacterial ribosomes respectively

Discussion

The E coli large subunit ribosomal protein bL12 con-tacts the CCA-arm of P-site bound tRNARecently we have applied an affinity labelling strategyto the human 80S ribosome whereby we have demon-strated that the lysyl residue 53 of the large subunitribosomal protein eL42 could covalently react withtRNAox at the PE hybrid site and that this reactioncompletely abolished the poly(Phe) synthesis activityof the human 80S ribosomes In this report applica-tion of the same affinity labelling strategy to E coliribosomes has led to the demonstration that the Ecoli rP bL12 contacts the CCA-end of P-site boundtRNA similarly to rP eL42 on the human 80S ribo-somes The bL12 protein had been localized within theE coli 70S ribosome by Cryo-electron Microscopy (29)as two dimers bound to one copy of protein uL10 (for-merly L10) which anchors the pentamer to the large

A

B C

Fig 5 Alignment of the amino acid sequences of the human eL42 and the E coli bL12 large subunit ribosomal proteins (A) comparison of fragment2100 of eL42 with fragment 17120 of bL12 The bottom line labels residues as either strictly conserved () highly conserved () or weaklyconserved () The alignment was generated with the program ClustalX The regions containing the GANK motif of bL12 or the GGQ motif ofeL42 are shown (in red and bold) (B) Alignment of the amino acid sequences of eL42 and bL12 in the region encompassing the GGQ motif ofeL42 Identical or chemically similar amino acid residues (in red) are labelled as either strictly conserved () or highly or weakly conserved (+)(C) Alignment of the amino acid sequences of eubacterial rPs bL12 in the region containing the GANK motif The strictly or strongly conservedLys or Arg residues including the labelled Lys-65 residue are shown in bold and green Other strictly or strongly conserved residues are shown inbold and blue All conserved residues are labelled with () Val is considered as isosteric with Thr

C Hountondji et al

8Downloaded from httpsacademicoupcomjbarticle-abstractdoi101093jbmvx0554082913Affinity-labelling-in-situ-of-the-bL12-protein-onby BIUSJ (Paris 6) useron 17 October 2017

50S ribosomal subunit This pentamer is called theribosomal bL12 lsquostalkrsquo It can be isolated either as anNH2-terminally acetylated 12 kDa polypeptide chainL7 or as a polypeptide with a free NH2 terminus L12both forms of the protein being currently named bL12The first ribosomal protein whose X-ray crystallo-graphic structure was solved is the CTD of proteinbL12 (30 31) Each monomer contains two distinctdomains linked by a flexible hinge (residues 3752)an elongated N-terminal domain (residues 136) thatis responsible for dimer interaction and binding touL10 and a large globular CTD (residues 53120)that interacts with elongation factors (30 31)

Identification of the residue of bL12 cross-linked insitu with tRNAox on E coli 70S ribosomesTo overcome the difficulty in identifying tRNAox-labelled peptides by mass spectrometry we have iden-tified indirectly the amino acid side chain(s) targetedby tRNAox by allowing this tRNA analogue to com-pete through the cross-linking reaction with PLP Wehave first identified the amino acid residue of bL12cross-linked with PLP Then we observed that prein-cubation of E coli 70S ribosomes with 1mM PLPprior to the addition of [32P]tRNAAspox preventedthe cross-linking reaction in situ between bL12 andtRNAox As the stoichiometry of cross-linking ofbL12 is one molecule of tRNAox or PLP covalentlybound per polypeptide chain the latter result suggeststhat both PLP and tRNAox might be cross-linked withthe e-amino group of the same lysyl residue of the pro-tein PLP has proven to be a suitable affinity label forlysyl residues near phosphate-binding sites (12 2022)In general formation of a Schiff base between the al-dehyde group of PLP and a lysyl e-amino group ispreceded by initial binding of the 50-phosphate group(12 2022) Conversion of the Schiff base to a stablesecondary amine is obtained by reduction with sodiumborohydride (NaBH4) Schiff base formation betweenthe aldehyde group of PLP and a lysyl e-amino groupof a protein is a mimicry of the mode of binding of thiscoenzyme to the catalytic lysyl residue through an aldi-mine (Schiff base) at the active site of transaminasesDuring the transamination reaction the transient formof the PLP bearing the amino group to be transferredonto a keto acid is PMP Therefore PLP is a usefultool to affinity labels the enzyme active sites containingcatalytic lysyl residues Previous chemical modificationin situ of E coli 50S ribosomal proteins by PLP (12)had led to the following observations in the 1980s (i)mainly seven ribosomal proteins were found to bemodified by PLP the bL12 protein being cross-linkedin majority (12) (ii) upon modification with PLP of the50S ribosomal subunit the interaction with the 30Sribosomal subunit the binding of the elongationfactor EF-G and the subsequent GTPase and trans-location activities were strongly impaired (iii) thepoly(U)-dependent poly(Phe) synthesis activity wascompletely lost (iv) the PLP-induced inhibition ofEF-G binding was shown to be due to the modificationof the bL12 protein (12) However the lysyl residue(s)cross-linked with PLP had never been identified In thepresent report taking advantage of the fact that the

bL12 protein was previously shown to behave similarlyon and off the ribosome (2931 3335) we have usedthe recombinant protein rather than the whole ribo-some to demonstrate the following (i) the E colibL12 protein can be cross-linked with PLP as previ-ously reported by the group of Gualerzi (12) (ii) thestoichiometry of cross-linking of PLP to the recombin-ant E coli bL12 protein is one molecule of PLP cova-lently bound per polypeptide chain (iii) the amino acidresidue of recombinant bL12 cross-linked with PLP isLys-65 (iv) when Lys-65 of the endogenous bL12 pro-tein of E coli 70S ribosomes was cross-linked in situwith PLP the 2030-dialdehyde derivative of tRNA(tRNAox) bound to the P-site could not cross-linkany more with this ribosomal protein This result sug-gests that both PLP and tRNAox are capable of cross-linking with the e-amino group of Lys-65 Howeverone cannot exclude that the failure of tRNAox tocross-link with the bL12 protein in the presence ofPLP is due to a steric hindrance that would preventthe tRNAox molecule from reaching its cross-linkingsite on bL12 Given that PLP is a small-size moleculewhich cannot span a wide region in the 3D structure ofthe protein another possibility is that the lysyl residuetargeted by tRNAox lies in proximity with Lys-65Lys-65 of E coli bL12 belongs to the large globularCTD (residues 53120) that interacts with tRNA andthe elongation factors

Possible common functional ribosomal or extra-ribo-somal roles for the L42 and L7L12 proteinsAn example of common functional role of the eL42and bL12 proteins on the ribosome concerns their in-volvement in the translocation step of translationelongation As discussed earlier we have previouslydemonstrated that the CCA end of a P-tRNA can becross-linked with the human large subunit ribosomalprotein eL42 This cross-link was shown to be specificfor a tRNA at the PE hybrid site as a tRNA in allother tRNA positions of pre-translocational ribosomescould not cross-link with any ribosomal protein (13)These data suggest that eL42 is involved in the trans-location step ie the movement of the bound tRNAfrom the P- to the E-site As for L7L12 it was previ-ously demonstrated in several studies that this riboso-mal protein is involved in the translocation of thepeptidyl-tRNA from the A- to the P-site (3840)The fact that the GANK and GGQTK motifsoccupy comparable positions in the 3D structures ofthe E coli bL12 and the human eL42 proteins respect-ively is compatible with their functional role in trans-location on the translating ribosomes (Fig 6) Finallya common functional role for Lys-53 of human eL42and Lys-65 of E coli bL12 on the ribosome is mostprobable because the former is strictly conserved (withvery few LysArg conservative replacements) in theamino acid sequences of all the rPs of the eL42family (13 27) while the latter is strictly conservedin the amino acid sequences of all eubacterial bL12(Fig 5C and (41)) Regarding the common extra-ribo-somal role of these proteins it was previously reportedthat eL42 is overexpressed in human hepatocellularcarcinoma as well as in several human tumour cell-

The E coli rP bL12 contacts the CCA-end of P-site tRNA

9Downloaded from httpsacademicoupcomjbarticle-abstractdoi101093jbmvx0554082913Affinity-labelling-in-situ-of-the-bL12-protein-onby BIUSJ (Paris 6) useron 17 October 2017

lines (42) Similarly an increased exposure of the in-testine to bacterial bL12 was previously observed inearly stage colorectal cancer patients (43) Thereforethe common extra-ribosomal role of the eukaryal eL42and the eubacterial bL12 proteins might be related totumour cell proliferation (42 43) It is generallybelieved that overexpression of components of thetranslation machinery in cancer cells could lead tothe discovery of new anticancer drugs An interestingexample in this respect is rapamycin which specificallyinactivates the mammalian target of rapamycin(mTOR) and had been shown to act as a powerfultumour-suppressive drug (44) Therefore eL42 andbL12 might represent targets of choice for anticancertherapy

Location of the E coli bL12 and the human eL42proteins in the 3D structures of the ribosomesAlong with the quaternary structure the most import-ant difference between human eL42 and E coli bL12 istheir location in the 3D structures of the ribosomesProtein bL12 the only multicopy component of theeubacterial ribosomes is located in the so-calledbL12 stalk (formerly L7L12 stalk) nearby the A-sitein sharp contrast to eL42 which would be located atthe E-site of eukaryotic ribosomes similarly to itscounterpart of the archaeon H marismortuiHowever it was reported that the CTD of each mono-mer of bL12 is highly mobile and capable of visitingthe functional A- P- and E-sites of the ribosomethanks to the marked flexibility of the whole protein(2830 45 46) Therefore it is not surprising that theCTD of the protein which is involved in the binding oftranslation factors and of the CCA-arm of tRNAwould be capable of contacting the CCA-end ofP-site bound tRNAox

Conclusion

In this report we have used tRNAox the 2030-dialde-hyde derivative of tRNA as a zero-length affinity label-ling reagent to demonstrate that the large subunitribosomal protein bL12 contacts the CCA-arm of P-

site bound tRNA on the E coli 70S ribosomes Thisreactive tRNA analogue was shown to fulfill all thecriteria for a site-directed affinity label of the bL12protein inside the ribosome (i) the stoichiometry ofcovalent binding of tRNAox to bL12 is one moleculeof tRNAox bound to one polypeptide chain of en-dogenous E coli bL12 in accordance with the presenceof one binding site for the CCA-arm of tRNA and forthe translation factors in the CTD of each of the foursubunits of the protein (ii) cross-linking in situ of bL12with tRNAox in the E coli 70S ribosomes provokesthe loss of the activity of poly(U)-dependent poly(Phe)synthesis which argues for a functional role of this rpin eubacterial ribosomes (iii) intact tRNA completelyprotected bL12 in the E coli 70S ribosomes againstcross-linking in situ with tRNAox suggesting a com-petition between native tRNAAsp and the dialdehydederivative thereof for the binding to the P-site BothtRNAox and PLP were shown to compete for the sameor for proximal cross-linking site(s) on bL12 inside theribosome Consistent with the latter observation thestoichiometry of cross-linking of PLP to the recombin-ant E coli bL12 protein is one molecule of PLP cova-lently bound per polypeptide chain similarly to thestoichiometry of tRNAox-labelling of the endogenousbL12 The amino acid residue of recombinant bL12cross-linked with PLP is Lys-65 Lys-65 of the62GANK65 motif of E coli bL12 matches with Lys-53 of the 49GGQTK53 motif of the large subunit ribo-somal protein eL42 which was previously shown tocross-link with P-site bound tRNAox on human 80Sribosomes in situ Finally E coli bL12 and humaneL42 proteins display significant primary structuresimilarities with an average of 4050 of identitiesand conservative replacements which argues for evo-lutionary conservation of these two proteins located atthe tRNA-CCA binding site on eubacterial andeukaryal ribosomes respectively

Supplementary Data

Supplementary Data are available at JB Online

Fig 6 Comparison of the 3D structure of Saccharomyces cerevisiae eL42 with that of the C-terminal factor binding domain of E coli bL12(A) C-terminal factor binding domain of E coli bL12 (PDB entry 1CTF) (B) S cerevisiae eL42 (PDB entry 4U4R chain q2) Secondarystructures a-helix (red) b-sheet (yellow) and coil (green) The GANK motif of bL12 and the GGQTK motif of eL42 are in stick representation(blue) The structures were visualized with pymol (httpswwwpymolorg)

C Hountondji et al

10Downloaded from httpsacademicoupcomjbarticle-abstractdoi101093jbmvx0554082913Affinity-labelling-in-situ-of-the-bL12-protein-onby BIUSJ (Paris 6) useron 17 October 2017

Acknowledgements

The authors would like to dedicate this article to a friend and col-league recently deceased Knud Nierhaus (Berlin) who contributedgreatly to our understanding of ribosomal translation We gratefullythank Dr Alain Brunelle for the mass spectrometric facilities andDrs Joel Pothier and Anne Woisard for fruitful discussions We aregrateful to Dr Suparna Sanyal for the kind gift of the strainBL21(DE3) carrying recombinant plasmid pET21L12C-6His

FundingThis work was supported by the West African Economic andMonetary Union (WAEMU UEMOA Ouagadougou BurkinaFaso grant no 02112013DDH-DSPSM-0239 to CH) We grate-fully thank Dr Cheikhe Hadjibou Soumare and Dr Guy-AmedeeAjanohoun for their constant interest for this work

Conflict of InterestNone declared

References

1 Nissen P Hansen J Ban N Moore P B and SteitzT A (2000) The structural basis of ribosome activity inpeptide bond synthesis Science 289 920930

2 Steitz T A and Moore P B (2003) RNA the firstmacromolecular catalyst the ribosome is a ribozymeTrends Biochem Sci 28 411418

3 Ban N Nissen P Hansen J Moore P B and SteitzT A (2000) The complete atomic structure of the largeribosomal subunit at 24 A resolution Science 289905920

4 Voorhees R M Weixlbaumer A Loakes D KelleyA C and Ramakrishnan V (2009) Insights into sub-strate stabilization from snapshots of the peptidyl trans-ferase center of the intact 70S ribosome Nat StructMol Biol 16 528533

5 Polikanov Y S Steitz T A and Innis C A (2014) Aproton wire to couple aminoacyl-tRNA accomodationand peptide-bond formation on the ribosome NatStruct Mol Biol 21 787793

6 Maracci C Wohlgemuth I and Rodnina M V (2015)Activities of the peptidyl transferase center of ribosomeslacking protein L27 Rna 21 16

7 Maden B E H and Monro R E (1968) Ribosome-catalyzed peptidyl transfer Effects of cations and pHvalue Eur J Biochem 6 309316

8 Baxter R M and Zahid N D (1978) The modificationof the peptidyl transferase activity of 50-S ribosomal sub-units LiCl-split proteins and L16 ribosomal protein byethoxyformic anhydride Eur J Biochem 91 4956

9 Wan K K Zahid N D and Baxter R M (1975) Thephotochemical inactivation of peptidyl transferase activ-ity Eur J Biochem 58 397402

10 Baxter R M White V T and Zahid N D (1980) Themodification of the peptidyl transferase activity of 50-Sribosomal subunits LiCl-split proteins and L16 riboso-mal protein by ethoxyformic anhydride Eur J Biochem110 161166

11 Tate W P Sumpter V G Trotman C N A HeroldM and Nierhaus K H (1987) The PeptidyltransferaseCentre of the Escherichia coli ribosome The histidine ofprotein L16 affects the reconstitution and control of theactive centre but is not essential for release-factor-mediated peptidyl-tRNA hydrolysis and peptide bondformation Eur J Biochem 165 403408

12 Ohsawa H Ohsawa E Giovane A and Gualerzi C(1983) Chemical modification in situ of Escherichia coli50S ribosomal proteins by the site-specific reagent pyri-doxal phosphate J Biol Chem 258 157162

13 Hountondji C Bulygin K Woisard A Tuffery PCrechet J B Pech M Nierhaus K H Karpova Gand Baouz S (2012) Lys53 of ribosomal protein L36ALand the CCA end of a tRNA at the PE hybrid site are inclose proximity on the human ribosome Chembiochem13 17911797

14 Ban N Beckmann R Cate J H D Dinman J DDragon F Ellis S R Lafontaine D L J Lindahl LLiljas A Lipton J M McAlear M A Moor P BNoller H F Ortega J Panse V G RamakrishnanV Spahn C M T Steitz T A Tchorzewski MTollervey D Warren A J Williamson J RWilson D Yonath A and Yusupov M (2014) Anew system for naming ribosomal proteins Curr OpinStruct Biol 24 165169

15 Parmeggiani A and Sander G (1981) Properties andregulation of the GTPase activities of elongation factorsTu and G and of initiation factor 2 Mol Cell Biochem35 129158

16 Matasova N B Myltseva S V Zenkova M AGraifer D M Vladimirov S N and Karpova G G(1991) Isolation of ribosomal subunits containing intactrRNA from human placenta estimation of functionalactivity of 80S ribosomes Anal Biochem 198 219223

17 Baouz S Woisard A Sinapah S Le Caer J PArgentini M Bulygin K Aguie G and HountondjiC (2009) The human large subunit ribosomal proteinL36A-like contacts the CCA end of P-site boundtRNA Biochimie 91 14201425

18 Kalogerakos T Hountondji C Berne P F DutkaS and Blanquet S (1994) Modification of aminoacyl-tRNA synthetases with pyridoxal-5rsquo-phosphateIdentification of the labeled amino acid residuesBiochimie 76 3344

19 Gillet S Hoang C B M Schmitter J M Fukui TBlanquet S and Hountondji C (1996) Affinity labelingof Escherichia coli histidyl-tRNA synthetase with react-ive ATP analogues identification of labeled amino acidresidues by matrix assisted laser desorption-ionizationmass spectrometry Eur J Biochem 241 133141

20 Hountondji C Fayat G and Blanquet S (1979)Complete inactivation and labeling of methionyl-tRNAsynthetase by periodate-treated initiator tRNA in thepresence of sodium cyanohydridoborate Eur JBiochem 102 247250

21 Hountondji C Blanquet S and Lederer F (1985)Methionyl-tRNA synthetase from Escherichia coli pri-mary structure at the binding site for the 3rsquo-end oftRNAfMet Biochemistry 24 11751180

22 Hountondji C Lederer F Dessen P and Blanquet S(1986) Escherichia coli tyrosyl- and methionyl-tRNAsynthetases display sequence similarity at the bindingsite for the 3rsquo-end of tRNA Biochemistry 25 1621

23 Hountondji C Schmitter J M Beauvallet C andBlanquet S (1987) Affinity labeling of Escherichia coliphenylalanyl-tRNA synthetase at the binding site fortRNA(Phe) Biochemistry 26 54335439

24 Hountondji C Schmitter J M Beauvallet C andBlanquet S (1990) Mapping of the active site ofEscherichia coli methionyl-tRNA synthetase identifica-tion of amino acid residues labeled by periodate-oxidizedtRNA(fMet) molecules having modified lengths at the 3rsquo-acceptor end Biochemistry 29 81908198

The E coli rP bL12 contacts the CCA-end of P-site tRNA

11Downloaded from httpsacademicoupcomjbarticle-abstractdoi101093jbmvx0554082913Affinity-labelling-in-situ-of-the-bL12-protein-onby BIUSJ (Paris 6) useron 17 October 2017

25 Baouz S Schmitter J M Chenoune L BeauvalletC Blanquet S Woisard A and Hountondji C (2009)Primary structure revision and active site mapping of Ecoli isoleucyl-tRNA synthetase by means of MALDImass spectrometry Open Biochem J 3 2638

26 Hountondji C Dessen P and Blanquet S (1986)Sequence similarities among the family of aminoacyl-tRNA synthetases Biochimie 68 10711078

27 Hountondji C Bulygin K Crechet J B Woisard ATuffery P Nakayama J Frolova L Nierhaus K HKarpova G and Baouz S (2014) The CCA-end of P-tRNA contacts both the human RPL42 and the A-sitebound translation termination factor eRF1 at the pepti-dyl tranferase center of the human 80S ribosome OpenBiochem J 8 5267

28 Dey D Oleinikov A V and Traut R R (1995) Thehinge region of Escherichia coli ribosomal protein L7L12 is required for factor binding and GTP hydrolysisBiochimie 77 925930

29 Montesano-Roditis L Glitz D G Traut R R andStewart P L (2001) Cryo-electron microscopic localiza-tion of protein L7L12 within the Escherichia coli 70Sribosome by difference mapping and nanogold labelingJ Biol Chem 276 1411714128

30 Leijonmarck M Eriksson S and Liljas A (1980)Crystal structure of a ribosomal component at 26 Aresolution Nature 286 824826

31 Leijonmarck M and Liljas A (1987) Structure of the C-terminal domain of the ribosomal protein L7L12 fromEscherichia coli at 17 A J Mol Biol 195 555579

32 Baouz S Woisard A Chenoune L Aguie G KeithG Schmitter J M Le Caer J P and Hountondji C(2009) Recent advances in the characterization of pepti-dyl transferase center zero-distance labeling of proteinsat or near the catalytic site of human 80S or E coli 70Sribosomes by means of periodate-oxidized tRNA Afr JBiochem Res 3 302311

33 Bocharov E V Gudkov A T and Arseniev A S(1996) Topology of the secondary structure elements ofribosomal protein L7L12 from E coli in solution FEBSLett 379 291294

34 Georgalis Y Dijk J Labischinski H and Wills P R(1989) The tetrameric form of ribosomal protein L7L12from Escherichia coli J Biol Chem 264 92109214

35 Ederth J Mandava C S Dasgupta S and Sanyal SA (2009) Single-step method for purification of activeHis-tagged ribosomes from a genetically engineeredEscherichia coli Nucleic Acids Res 37 19

36 Annan R S and Carr S A (1996) Phosphopeptideanalysis by matrix-assisted laser desorption time-of-flight mass spectrometry Anal Chem 68 34133421

37 Kothe U Wieden H J Mohr D and Rodnina M V(2004) Interaction of helix D of elongation factor Tuwith helices 4 and 5 of protein L7L12 on the ribosomeJ Mol Biol 336 10111021

38 Savelsbergh A Mohr D Kothe U Wintermeyer Wand Rodnina M V (2005) Control of phosphate releasefrom elongation factor G by ribosomal protein L7L12EMBO J 24 43164323

39 Diaconu M Kothe U Schlunzen F Fischer NHarms J M Tonevitsky A G Stark H RodninaM V and Wahl M C (2005) Structural basis for thefunction of the ribosomal L7L12 stalk in factor bindingand GTPase activation Cell 121 9911004

40 Wahl M C Bourenkov G P Bartunik H D andHuber R (2000) Flexibility conformational diversityand two dimerization modes in complexes of ribosomalprotein L12 EMBO J 19 174186

41 Wieden H J Wintermeyer W and Rodnina M V(2001) A common structural motif in elongation factorTs and ribosomal protein L7L12 may be involved in theinteraction with elongation factor Tu J Mol Evol 52129136

42 Kim J H You K R Kim I H Cho B H Kim CY and Kim D G (2004) Over-expression of the ribo-somal protein L36a gene is associated with cellularproliferation in hepatocellular carcinoma Hepatology39 129138

43 Boleij A Roelofs R Schaeps R M J Schulin TGlaser P Swinkels D W Kato I and Tjalsma H(2010) Increased exposure to bacterial antigen rpL7L12in early stage colorectal cancer patients Cancer 11640144022

44 Shenoy N Kessel R Bhagat T D Bhattacharyya SYu Y McMahon C and Verma A (2012) Alterationsin the ribosomal machinery in cancer and hematologicdisorders J Hematol Oncol 5 19

45 Dey D Bochkariov D E Jokhadze G G and TrautR (1998) Cross-linking of selected residues in the N- andC-terminal domains of Escherichia coli protein L7L12 toother ribosomal proteins and the effect of elongationfactor Tu J Biol Chem 273 16701676

46 Bocharov E V Gudkov A T Budovskaya E V andArseniev A S (1998) Conformational independence ofN- and C-domains in ribosomal protein L7L12 and inthe complex with protein L10 FEBS Lett 423 347350

C Hountondji et al

12Downloaded from httpsacademicoupcomjbarticle-abstractdoi101093jbmvx0554082913Affinity-labelling-in-situ-of-the-bL12-protein-onby BIUSJ (Paris 6) useron 17 October 2017

25 Baouz S Schmitter J M Chenoune L BeauvalletC Blanquet S Woisard A and Hountondji C (2009)Primary structure revision and active site mapping of Ecoli isoleucyl-tRNA synthetase by means of MALDImass spectrometry Open Biochem J 3 2638

26 Hountondji C Dessen P and Blanquet S (1986)Sequence similarities among the family of aminoacyl-tRNA synthetases Biochimie 68 10711078

27 Hountondji C Bulygin K Crechet J B Woisard ATuffery P Nakayama J Frolova L Nierhaus K HKarpova G and Baouz S (2014) The CCA-end of P-tRNA contacts both the human RPL42 and the A-sitebound translation termination factor eRF1 at the pepti-dyl tranferase center of the human 80S ribosome OpenBiochem J 8 5267

28 Dey D Oleinikov A V and Traut R R (1995) Thehinge region of Escherichia coli ribosomal protein L7L12 is required for factor binding and GTP hydrolysisBiochimie 77 925930

29 Montesano-Roditis L Glitz D G Traut R R andStewart P L (2001) Cryo-electron microscopic localiza-tion of protein L7L12 within the Escherichia coli 70Sribosome by difference mapping and nanogold labelingJ Biol Chem 276 1411714128

30 Leijonmarck M Eriksson S and Liljas A (1980)Crystal structure of a ribosomal component at 26 Aresolution Nature 286 824826

31 Leijonmarck M and Liljas A (1987) Structure of the C-terminal domain of the ribosomal protein L7L12 fromEscherichia coli at 17 A J Mol Biol 195 555579

32 Baouz S Woisard A Chenoune L Aguie G KeithG Schmitter J M Le Caer J P and Hountondji C(2009) Recent advances in the characterization of pepti-dyl transferase center zero-distance labeling of proteinsat or near the catalytic site of human 80S or E coli 70Sribosomes by means of periodate-oxidized tRNA Afr JBiochem Res 3 302311

33 Bocharov E V Gudkov A T and Arseniev A S(1996) Topology of the secondary structure elements ofribosomal protein L7L12 from E coli in solution FEBSLett 379 291294

34 Georgalis Y Dijk J Labischinski H and Wills P R(1989) The tetrameric form of ribosomal protein L7L12from Escherichia coli J Biol Chem 264 92109214

35 Ederth J Mandava C S Dasgupta S and Sanyal SA (2009) Single-step method for purification of activeHis-tagged ribosomes from a genetically engineeredEscherichia coli Nucleic Acids Res 37 19

36 Annan R S and Carr S A (1996) Phosphopeptideanalysis by matrix-assisted laser desorption time-of-flight mass spectrometry Anal Chem 68 34133421

37 Kothe U Wieden H J Mohr D and Rodnina M V(2004) Interaction of helix D of elongation factor Tuwith helices 4 and 5 of protein L7L12 on the ribosomeJ Mol Biol 336 10111021

38 Savelsbergh A Mohr D Kothe U Wintermeyer Wand Rodnina M V (2005) Control of phosphate releasefrom elongation factor G by ribosomal protein L7L12EMBO J 24 43164323

39 Diaconu M Kothe U Schlunzen F Fischer NHarms J M Tonevitsky A G Stark H RodninaM V and Wahl M C (2005) Structural basis for thefunction of the ribosomal L7L12 stalk in factor bindingand GTPase activation Cell 121 9911004

40 Wahl M C Bourenkov G P Bartunik H D andHuber R (2000) Flexibility conformational diversityand two dimerization modes in complexes of ribosomalprotein L12 EMBO J 19 174186

41 Wieden H J Wintermeyer W and Rodnina M V(2001) A common structural motif in elongation factorTs and ribosomal protein L7L12 may be involved in theinteraction with elongation factor Tu J Mol Evol 52129136

42 Kim J H You K R Kim I H Cho B H Kim CY and Kim D G (2004) Over-expression of the ribo-somal protein L36a gene is associated with cellularproliferation in hepatocellular carcinoma Hepatology39 129138

43 Boleij A Roelofs R Schaeps R M J Schulin TGlaser P Swinkels D W Kato I and Tjalsma H(2010) Increased exposure to bacterial antigen rpL7L12in early stage colorectal cancer patients Cancer 11640144022

44 Shenoy N Kessel R Bhagat T D Bhattacharyya SYu Y McMahon C and Verma A (2012) Alterationsin the ribosomal machinery in cancer and hematologicdisorders J Hematol Oncol 5 19

45 Dey D Bochkariov D E Jokhadze G G and TrautR (1998) Cross-linking of selected residues in the N- andC-terminal domains of Escherichia coli protein L7L12 toother ribosomal proteins and the effect of elongationfactor Tu J Biol Chem 273 16701676

46 Bocharov E V Gudkov A T Budovskaya E V andArseniev A S (1998) Conformational independence ofN- and C-domains in ribosomal protein L7L12 and inthe complex with protein L10 FEBS Lett 423 347350

C Hountondji et al

12Downloaded from httpsacademicoupcomjbarticle-abstractdoi101093jbmvx0554082913Affinity-labelling-in-situ-of-the-bL12-protein-onby BIUSJ (Paris 6) useron 17 October 2017