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Biochimie 158 (2019) 20e33

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Biochimie

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Research paper

Ribosomal protein eL42 contributes to the catalytic activity of theyeast ribosome at the elongation step of translation

Codjo Hountondji a, *, Jean-Bernard Cr�echet a, Mayo Tanaka b, Mieko Suzuki c,Jun-ichi Nakayama b, c, **, Blanche Aguida a, Konstantin Bulygin a, d, Jean Cognet e,Galina Karpova d, Soria Baouz a

a Sorbonne Universit�e, Campus Pierre et Marie Curie, Laboratoire “Enzymologie de l’ARN”, SU-UR6, (Batiment B), Case courrier 60 - 4, Place Jussieu, F-75252, Paris Cedex 05, Franceb Division of Chromatin Regulation, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki, 444-8585, Japanc Graduate School of Natural Sciences, Nagoya City University, 1 Yamanohata, Mizuho, Nagoya, Aichi, 467-8501, Japand Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr Lavrentieva, 8, 630090, Novosibirsk,Russiae Sorbonne Universit�e, Campus Pierre et Marie Curie, Laboratoire Jean Perrin, CNRS-UMR8237, (Tour 32), 4, Place Jussieu, F-75252, Paris Cedex 05, France

a r t i c l e i n f o

Article history:Received 2 October 2018Accepted 8 December 2018Available online 11 December 2018

Keywords:eL42 protein from human orSchizosaccharomyces pombethe GGQ motif of eL42Lys-55 of S. pombe eL42Eukaryal 80S ribosomesMechanism of peptidyl transfer andpeptidyl-tRNA hydrolysis by eukaryotic 80SribosomesThe peptidyl transferase center of 80Sribosomes from eukaryotes

Abbreviations: ß-ME, ß-mercaptoethanol; DTT, diarchaeal large subunit ribosomal protein L15 (formerleL42, eukaryal or archaeal large subunit ribosomal pL42AB in yeast, L36a or L36a-like in human, or L44eminimal medium; Hma, Haloarcula marismortui; PTCPBS, phosphate-buffered saline; rpl42þ, the gene coTCA, trichloroacetic acid; tRNAox, periodate-oxidizederivative of tRNA; uL15, universal large subunit riboL15 in bacteria, L28 in yeast or L27a in mammals).* Corresponding author. Sorbonne Universit�e, Cam

Laboratoire SU-UR6 ‘‘Enzymologie de l’ARN’’ (Bat. BSaint-Bernard, F-75251, Paris Cedex 05, France.** Corresponding author. Division of Chromatin RegBasic Biology, Nishigonaka 38, Myodaiji, Okazaki, 444

E-mail addresses: codjo.hountondji@upmc.fr (C. Hac.jp (J.-i. Nakayama).

https://doi.org/10.1016/j.biochi.2018.12.0050300-9084/© 2018 Elsevier B.V. and Société Française

a b s t r a c t

The GGQ minidomain of the ribosomal protein eL42 was previously shown to contact the CCA-arm of P-site bound tRNA in human ribosome, indicating a possible involvement of the protein in the catalyticactivity. Here, using Schizosaccharomyces pombe (S. pombe) cells, we demonstrate that the GGQ mini-domain and neighboring region of eL42 is critical for the ribosomal function. Mutant eL42 proteinscontaining amino acid substitutions within or adjacent to the GGQ minidomain failed to complement thefunction of wild-type eL42, and expression of the mutant eL42 proteins led to severe growth defects.These results suggest that the mutations in eL42 interfere with the ribosomal function in vivo.Furthermore, we show that some of the mutations associated with the conserved GGQ region lead toreduced activities in the poly(Phe) synthesis and/or in the peptidyl transferase reaction with respect topuromycin, as compared with those of the wild-type ribosomes. A pK value of 6.95 was measured for theside chain of Lys-55/Arg-55, which is considerably less than that of a Lys or Arg residue. Altogether, ourfindings suggest that eL42 contributes to the 80S ribosome's peptidyl transferase activity by promotingthe course of the elongation cycle.

© 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rightsreserved.

thiotreitol; eL15, eukaryal ory L15 in yeast and in human);rotein L42 (formerly L42A orin archaea); EMM, Edinburgh, peptidyl transferase center;ding for the wild-type eL42;d tRNA, the 20 ,30-dialdehydesomal protein L15 (formerly

pus Pierre et Marie Curie,), Case courrier 60 - 7, Quai

ulation, National Institute for-8585, Japan.ountondji), jnakayam@nibb.

de Biochimie et Biologie Molécul

1. Introduction

The ribosome is responsible for protein biosynthesis in allkingdoms of life. It is one of the most complex supramolecularstructures known in any cell. It is composed of two subunits ofunequal size. The large subunit (50S in eubacteria and arch-aeabacteria, and 60S in eukaryotes) contains the active site, thepeptidyl transferase center (PTC), which catalyzes the formation ofpeptide bonds in the growing polypeptide, while the small subunit(30S in bacteria and 40S in eukaryotes) carries the decoding sitewhere anmRNA codon is recognized by the anticodon of its cognateaminoacyl-tRNA. All the ribosomal proteins (rps) mentioned in thepresent report were called according to the new system for namingribosomal proteins [1]. For example, the eukaryal or archaeal largesubunit ribosomal proteins of the L44e family (formerly ribosomal

aire (SFBBM). All rights reserved.

C. Hountondji et al. / Biochimie 158 (2019) 20e33 21

protein L42 in yeast, L36a in human, or L44e in archaea) are calledeL42 [1].

The current view of ribosomal peptidyl transfer is that theribosome is a ribozyme and that ribosomal proteins are notinvolved in catalysis of peptide bond formation [2e4]. Historically,this view stemmed from the fact that the three-dimensionalstructure of the 50S ribosomal subunit of the archaeon Haloarculamarismortui (Hma) reveals a void of protein electron density in aradius of 18 angstr€oms of the PTC [2,5]. However, this proposal isstill a matter of controversy, and the catalytic mechanism of pep-tide bond formation is still not fully understood inmolecular terms.For example, an initial assumption concerning the involvement ofN3 atom of the universally conserved E. coli A2451 (A2486 in Hma)[2] in the peptidyl transfer reaction for a general acid/base catalyticmechanism has been finally judged unlikely because the activity ofthe ribosomewas unchangedwhen A2451 (A2486) was replaced byany other nucleotide in the E. coli 70S ribosome [6,7]. The univer-sally conserved E. coli A2602 has also been proposed to be involvedin the release factor-mediated peptidyl-tRNA hydrolysis [8,9].However, other experiments have revealed that an abasic 2602residue in 23S rRNA had no effect on the reaction of peptide release,so that a direct involvement of A2602 in catalysis could be excluded[10]. Recently, the group of Steitz has proposed an alternate modelfor peptide bond formation involving the N-terminus of Thermusthermophilus ribosomal protein bL27 [1,11]. In fact, crystallographicdata on the full 70S ribosome from T. thermophilus show that bL27extends with its N-terminal tail into the PTC and makes contactwith the tRNA substrates [12]. However, it has been recentlydemonstrated that bL27 [1] does not play any significant role inpeptide bond formation [13]. Moreover, ribosomes from archaea oreukaryotes do not have bL27 or any homologous counterpart,indicating that it cannot be a part of an evolutionarily conservedpeptidyl transfer mechanism, which is expected to rely on the sameresidues in all organisms [12,14].

Recent cross-linking studies have shed light on the structuralaspects of molecular interactions between tRNA and the eukaryote-specific large subunit ribosomal protein eL42 from mammals[15e17] that had been previously proposed to be implicated inpeptidyl transfer [18]. This protein is strongly conserved in eu-karyotes and archaea. It belongs to the former L44e family of ri-bosomal proteins, a representative of which is Hma eL42. The largeribosomal subunit of this archaeon had been analyzed by X-raycrystallography [2,5]. According to the crystallographic data, eL42from the archaeon Hma is located at the large ribosomal subunit E-site, where it interacts with the two unstacked cytosine residuesC75 and C74 of a native tRNA through an extension loop of theprotein [19]. This protein has also been shown to contain sevenmonomethylated residues, among which is the glutaminyl residue(Q51) contained in the conserved GGQ motif of the eL42 in alleukaryotic 80S ribosomes [16]. This motif is identical to the uni-versally conserved GGQ motif common for all class-1 translationtermination factors responsible for stop codon recognition and fortriggering hydrolysis of the complex ester bond between the pep-tidyl moiety and the 30-terminal ribose of the P site-bound pep-tidyl-tRNA [20e23]. However, the question of the functional role ofthe GGQmotif of the eL42 has never been addressed. The possibilitythat this protein might be involved in the catalytic activity of 80Sribosomes providing peptidyl transfer is one of the subjects of thisstudy. Since, to date, structural and biochemical studies failed todetermine PTC at the molecular level, our hope was that the widevariety and the clear-cut answers of the genetic approaches avail-able in the yeast system should provide insights into the molecularmechanism of the peptidyl transfer reaction through the identifi-cation of the structural or functional roles of specific amino acidresidues in the eL42 protein. Here, we demonstrate that expression

of mutant eL42 containing amino acid changes within or adjacentto the GGQ motif in S. pombe cells lead to severe growth defect.Furthermore, we present evidence that ribosomes prepared fromS. pombe cells expressing mutant eL42 displayed impaired activitiesin the poly(U)-dependent poly(Phe) synthesis and/or in the pep-tidyl transfer, as compared with those of the wild-type cells. Thecross-linking in situ of tRNA bearing oxidized 30-terminal ribose(tRNAox) to eL42 in the yeast 80S ribosomes as a function of the pHof the incubation mixture is shown to display a pK value of about6.95 for the side chain of Lys55 (or Arg55) in both thewild-type andthe Q53K, K55R or P56Q eL42 mutants. The data obtained bringnew insights into the molecular mechanisms providing translationin eukaryotes, highlighting the contribution of eL42 to the catalyticactivity of the ribosome.

2. Materials and methods

2.1. Strains and plasmids

All of the yeast strains were grown at 30 �C in YEA (0.5% yeastextract, 3% glucose, 75 mg adenine) or Edinburgh minimal medium(EMM) supplemented with amino acids for auxotrophic markers.To obtain strains expressing mutant Rpl42/eL42, the rpl42þ-codingsequence (�456~þ782) was PCR-amplified from S. pombe genomicDNA and cloned into pCRII vector using the TOPO-TA cloning kit(Invitrogen). Site-directed mutagenesis was used to introduceamino acid substitutions. Mutated rpl42þ-coding region wassubcloned into KS-ura4 vector using BamHI and EcoRI restrictionsites. The resultant plasmids were cleaved by MfeI and introducedinto the original rpl42þ locus. To replace the wild-type rpl42þ allelewith the mutant rpl42 allele, strains that had lost the ura4þ genethrough internal homologous recombination were isolated usingcounter-selective medium containing FOA, and the replaced allelewas confirmed by sequencing PCR-amplified genomic DNA.

To express mutant Rpl42/eL42 ectopically, rpl42þ-coding region(þ1~þ511) containing each mutation was PCR-amplified andcloned into the pCRII vector using the TOPO-TA cloning kit (Invi-trogen). The rpl42þ-coding region was then subcloned into pREP1plasmid using NdeI and BamHI sites, and the resultant plasmidswere introduced into wild-type S. pombe strain (JY741). Trans-formed cells were selected on EMM containing 16 mM thiamine.After inoculating for several days on the same EMM medium, thetransformed cells were streak onto EMM without thiamine to ex-press mutant Rpl42/eL42.

2.2. Translation factors and tRNA

Purified elongation factors from calf brain EF-1a, EF-1b, EF-2were obtained as described in Ref. [24]. tRNAAsp was purified aspreviously described [15]. Poly(U), puromycin and tRNAPhe from E.coliwere from Sigma-Aldrich and L-[14C(U)]Phenylalanine (18 GBq/mmol) or [3H]Phenylalanine (4740 GBq/mmol) from PerkinElmer.tRNAPhe was aminoacylated using [14C(U)]Phenylalanine or [3H]Phenylalanine with an excess amount of partially purifiedphenylalanyl-tRNA synthetase from E. coli [25]. [3H]Phe-tRNAPhe

(128 GBq/mol) was acetylated following the method described byHaenni and Chapeville [26].

2.3. Purification of ribosomes and ribosomal proteins fromSchizosaccharomyces pombe

Yeast S. pombe strains were grown in YEA medium at 28 �C andcollected by centrifugation at a cell density of 4 A600. The pellet waswashed with phosphate-buffered saline (PBS) and resuspended inlysis buffer containing 25mM Tris-HCl [pH 7.5], 10mM MgCl2,

C. Hountondji et al. / Biochimie 158 (2019) 20e3322

50mM NH4Cl, 1mM dithiotreitol (DTT) and one tablet mini EDTA-free protease inhibitor cocktail (Roche Diagnostics). S. pombe cellswere disrupted using microfluidizer from Microfluidics after 3passes at 30,000 psi using the G10Z chamber (87 microns). Theextract was cleared by centrifugation for 30min at 28,000�g. Su-pernatant was treated with 20 mg/ml DNase and centrifuged at200,000�g for 3 h in a 70 Ti rotor (Beckman) The ribosomal pelletwas resuspended in buffer containing 25mM Tris-HCl [pH 7.5],10mMMgCl2, 50mMNH4Cl and 7mM ß-mercaptoethanol (ß-ME).After centrifugation at 23,000�g for 20min, puromycin (0.5mM)and GTP (0.5mM) were added to the supernatant, and the mixturewas incubated at 37 �C for 10min. Purified ribosomes were thenobtained by two centrifugations at 200,000�g for 3 h through an18% sucrose layer containing 50mM Tris-HCl [pH 7.5], 5mM MgCl2and 0.5M KCl, and kept at �30 �C after resuspension and dialysisagainst buffer containing 25mM Tris-HCl [pH 7.5], 10mM MgCl2,50mM NH4Cl, 50% glycerol, and 1mM DTT. The S. pombe recom-binant eL42 protein was purified as previously described [17]. TheeL42-D(GGQTK) mutant (i.e. the S. pombe eL42 protein whoseGGQTK motif has been deleted) and the single K55Q mutant(named eL42-K55Q) were purified as described recently [27].

2.4. Purification of EF-3

TKBL40 (BL21 Lex His-Tagged eEF-3 pET IIa) was kindly giftedfrom Maria Mateyak (Rutgers RWJ MED School, Biochemistry andmolecular Biology department, Piscataway, US). A fresh overnightsaturated culture was used to inoculate 2 liters of LB with 50 mg/mlampicillin and grownwith shaking at 37 �C to 0.6 A600, after whichinduction with 0.2mM isopropyl-ß-D-thiogalacto-pyranoside tookplace at 23 �C for 15 h. After harvest, the cells (14 g) were washed inPBS, sonicated 15 times for 10 secondes at 4 �C in 40ml buffer B(25mM Tris-HCl pH7.5, 0.5M NaCl, 10mM imidazole, and 1mMß-ME) containing, 1mM MgCl2, 0.5mg/ml lysosyme, 100 mg/mlDNase and one tablet of complete mini EDTA-free protease inhib-itor cocktail (Roche Diagnostics), and centrifuged (120,000�g for30min). The supernatant was applied two times on 1ml HisgraviTrap column (GE Healthcare) equilibrated in buffer B. Afterwashingwith 20ml buffer A containing 20mM imidazole, EF-3 waseluted in 10ml buffer B with 100mM imidazole. This fraction wasconcentrated in an Amicon ultrafiltration apparatus (Milliporemembrane PM10), dialysed against buffer containing 25mM Tris-HCl [pH7.5], 1mM MgCl2, 50mM NH4Cl, 1mM DTT, 50% glyceroland stored at �30 �C.

2.5. Poly(U)-dependent poly(Phe) synthesis activity

Poly(Phe) synthesis was determined as incorporation of L-[14C(U)]Phenylalanine into hot trichloroacetic acid (TCA)-insolublematerial as previously described [24]. The reaction mixture (100 ml)contained 40mM Tris-HCl [pH7.5], 7mM MgCl2, 80mM NH4Cl,1mMDTT,1mMATP,1mM Phosphoenolpyruvate, 0.3mM creatinephosphate, 0.5mM GTP, 50 mg/ml pyruvate kinase, 50 mg/ml crea-tine kinase, 5 mM tRNAPhe (first charged during a 30min incubationat 30 �C with a 2 fold excess of L-[14C(U)]Phenylalanine [5 GBq/mmol] and a saturating amount of partially purified phenylalanyl-tRNA synthetase), 3.5 mg poly(U), 0.5 mM EF-1a, 0.15 mM EF-1b,0.35 mM EF-2, 1 mM EF-3, and 0.4 mM S. pombe 80S ribosomes.During incubation at 30 �C, 30 ml aliquots were withdrawn at timesindicated, spotted on glass fiber filters and hot TCA insolubleradioactivity was determined.

2.6. Peptidyl transferase activity

Ac-[3H]Phe-tRNA.poly(U).S.pombe ribosome complexes were

formed during incubation for 15min at 30 �C in 100 ml containing2 mM S. pombe ribosome, 3 mM Ac-[3H]Phe-tRNA(128 GBq/mol),20 mg poly(U) in 25mM Tris-HCl [pH 7.5], 10mM MgCl2, 30mMNH4Cl, 30mM KCl, 1mM DTT (buffer A) and separated on superose12 HR 10/30 column (GE Healthcare) equilibrated in the samebuffer. The isolated complexes were concentrated to 200 ml usingmicrocon 100 (Amicon) and the concentration of the complexesdetermined (One A260 unit of Ac-aa-tRNA.mRNA.ribosome complexwas assumed to contain 24 pmol 80S.complex). Peptidyl trans-ferase activity was determined in 72 ml reaction mixture containingin buffer A 0.4 mM Ac-Phe-tRNA.poly(U).ribosome complex, 0.8 mMeEF-3, 0.5 mMGTP, 0.5 mMATP in the presence or absence of 1.5mMPuromycin. After 15min incubation at 30 �C, 68 ml were withdrawnand placed on Whatman GFC filters. The filters were washed withcold TCA and dried before radioactivity counting. The extent ofpuromycin reactivity (PTC activity) was determined by comparingthe radioactivity of the unreacted acid-insoluble material in thepresence and in the absence of the antibiotic.

2.7. Phe-tRNAPhe binding to the A-site

Enzymatic binding of [3H]Phe-tRNAPhe to the A-site of poly(U)-programmed wild-type or mutant S. pombe ribosomes was per-formed as in Ref. [24]. Aliquots (45 ml) containing 40mM Tris-HCl[pH 7.5], 7mM MgCl2, 80mM NH4Cl, 1mM DTT, 0.5mM GTP,0.2 mM EF-1b, and 0.5 mM [3H]Phe-tRNAPhe (specific activity, 85GBq/mmol) were preincubated for 10min at 37 �C in the presenceor absence of 0.6 mM EF-1a before the reactionwas started with theaddition of poly(U)-programmed ribosomes (15 ml) at final con-centrations of 0.3 mM 80S ribosomes and 4.2 mg of poly(U). Theenzymatic binding mediated by EF-1a and non-enzymatic bindingof [3H]Phe-tRNAPhe to the ribosomal A-site was determined after6min at 37 �C by the nitrocellulose binding assay.

2.8. Cross-linking of tRNAox to 80S ribosomes or to the purifiedrecombinant S. pombe eL42 protein

Cross-linking of tRNAox to yeast 80S ribosomes was performedas described earlier [15e17]. Reaction mixtures (20 ml each) con-tained 10 pmol of 80S ribosomes, 100 pmol of oligoribonucleotideGAA-GAC-UAA-AAA as mRNA and 20 pmol of [32P]tRNAAspox inbuffer A (50mM Tris-HCl at the indicated pH, 100mM KCl, 10mMMgCl2 and 0.5mM EDTA) with incubation for 40min at 25 �C. Forthe cross-linking studies as a function of the pH of the incubationmixture, 5mM NaBH3CN was used as reducing agent instead ofNaBH4. Cross-linked proteins were analyzed by SDS-PAGE on 10%polyacrylamide gels, and the resulting gels were subjected toautoradiography. The cross-linking yields were calculated asdescribed earlier [17]. Cross-linking of tRNAox to the purified re-combinant S. pombe eL42 protein or to the eL42-D(GGQTK) or eL42-K55Q variants was performed as previously described [15e17].

3. Results

3.1. Conserved amino acids within or adjacent to the conservedGGQ motif of eL42 are critical for its function in the S. pombe cells

We have previously demonstrated that the lysyl residue 53 ofthe human large subunit ribosomal protein eL42 could be cross-linked with tRNAox at the P/E hybrid site, and that this covalentreaction completely abolished the poly(Phe) synthesis activity ofthe human 80S ribosomes, suggesting that Lys-53 might play afunctional role [16]. Using S. pombe, we have also demonstratedthat the corresponding Lys-55 residue of yeast eL42 is post-translationally methylated in cells and that the methylation state is

C. Hountondji et al. / Biochimie 158 (2019) 20e33 23

critical to ribosomal function and cell proliferation control [28].Methylation at this lysyl residue has also been identified in otherspecies, including Saccharomyces cerevisiae [29], plants [30] andhuman [16,28], implying its functional importance in ribosomalfunction. This lysyl residue is located next to the highly conservedGGQ motif (Fig. 1) that is identical to the universal GGQ motifpresent in all class-1 translation termination factors, although itsfunctional implication has yet to be determined in the eL42 familyof ribosomal proteins.

To investigate the role of the conserved motif of eL42 in itsfunction, we have introduced mutant rpl42 gene (S. pombe geneencoding eL42) into original rpl42þ locus with an auxotrophicmarker gene and tried to replace wild-type rpl42þ with each ofmutant alleles (seeMaterials andMethods).We observed, however,that the wild-type rpl42þ could not be replaced by a mutant rpl42gene with six amino acids deletion (51-GGQTKP-56) (Fig. 2A). Thisresult would suggest that this motif is critical for the eL42 function.

To identify the amino acid residue(s) related to this effect, we

Fig. 1. Conserved region in the GGQ minidomain of the eukaryotic and archaeal eL42 prregion encompassing the GGQ motif common to three groups of proteins: the class-1 tranarchaeal organisms and proteins exhibiting peptidyl-tRNA hydrolase activity. The amino acidred. The bottom line concerns all the groups except the one of the archaeal eL42 proteins (lochighly conserved (:). (*, black star) designates the group of archaeal eL42 proteins. The alig

have introduced a series of amino acid substitutions in the rpl42gene and examined whether wild-type rpl42þ could be replaced byeach of the mutant genes by the same replacement method. Whilewe succeeded in isolating cells expressing mutant rpl42 for G51R,G51A, or Q53K, we failed to obtain cells for G49R, G49A, G52R,G52A, K55Q, or K55E (Fig. 2A). These results suggest that glycylresidues 49 and 52, as well as lysyl residue 55 are critical for theeL42 function.

3.2. Expression of mutant eL42 containing amino acid changeswithin or adjacent to the conserved GGQ motif of eL42 results ingrowth defects in S. pombe cells

First attempts to demonstrate the role of the GGQmotif showedthat some of the S. pombe cells harboring both wild-type andmutant rpl42 genes with amino acid substitutions within or adja-cent to the GGQ motif display growth defects (data not shown).These results strongly suggest that the mutant proteins exhibited a

oteins, and the translation termination factors. Multiple sequence alignment of theslation termination factors, the rPs of the eL42 family from different eukaryotic andresidues that are conserved in majority in at least one of the three groups are colored

ated at the top of the figure). It labels residues as either strictly conserved (*, red star) ornment was generated with the program ClustalX.

Fig. 2. Amino acid changes within or adjacent to the GGQ minidomain of eL42 result in growth defects in the yeast Schizosaccharomyces pombe. (A), Summary of thereplacement experiments using conventional gene replacement method and dominant negative growth defect by mutant eL42 proteins. Viability (O) or lethality (X) weredetermined according to the gene replacement method. The poly(U)-dependent poly(Phe) synthesis and peptidyl transferase activities of S. pombe mutant ribosomes, as deducedfrom Fig. 3, were expressed each as a percent of the activity of the wild-type ribosome considered as 100%. When a mutant strain was not viable, activity measurement wasimpossible and irrelevant (�). Strains expressing mutant eL42 with K55R* or P56Q* were previously described [28]. (B), Dominant-negative growth defects by expressing mutanteL42 proteins. S. pombe cells containing an ectopic plasmid with wild-type or mutant rpl42 gene were isolated on a minimal medium lacking leucine, and the effect of overexpressedwild-type and mutant eL42 proteins were tested by streaking on a minimal medium with (induction -) or without out thiamine (induction þ). S. pombe cells without ectopicplasmid were used as the control.

C. Hountondji et al. / Biochimie 158 (2019) 20e3324

dominant negative effect, indicating that some amino acid residuesin this region are critical to eL42 function. To confirm the dominantnegative effect and, at the same time, to gain further insight into therole of the GGQ motif and of neighboring residues, we expressedmutant eL42 proteins from an ectopic plasmid in wild-type cellsand examined the effect on their growth (Fig. 2B). Mutant rpl42

genes were repressed in medium containing thiamine (induction-), but overexpressed when the culture was shifted to mediumlacking thiamine (induction þ). The yeast cells expressing D51-56,G49R, G52R, or G52Amutants showed severe growth defects, whilea moderate growth defect was seen for the G49A, G51R, G51A,Q53K, or K55Q mutant (Fig. 2B). Even though this approach is less

C. Hountondji et al. / Biochimie 158 (2019) 20e33 25

demonstrative than that of the gene replacement method, it isinteresting to note that substitution of glycyl residues 49 and 52 inS. pombe eL42 (G49R, G52A and G52R mutants) provoked severegrowth defects (Fig. 2B), in accordance with the failure of genereplacement attempt (Fig. 2A). However, the fact that only a mod-erate growth defect was observed for the G49A or K55Q mutant islikely to be due to the presence of traces of the wild-type eL42protein inherent to this type of approach. Altogether, these resultsconfirm the aforementioned dominant negative effect and suggestthat glycyl residues 49 and 52, as well as lysyl residue 55 are criticalfor the eL42 function. Moreover, the data in Fig. 2A and B areremarkably comparable to those previously reported by severalresearch groups on the mutational analysis of the GGQminidomainof the eukaryotic and the prokaryotic release factors (Table S1 and[20,22,31e34]). It is seen that the substitutions of central glycine inthe GGQ motif with alanine or arginine in eL42 S. pombe provokedlethality or severe growth defect (Fig. 2A and B), and the samesubstitutions in release factors resulted in lethality or a decrease ofthe constant rate for peptide release by four orders of magnitude,respectively (Table S1). Similarly, except for the yeast eRF1 (Table S1and [31]), substitutions of the glutamine residue of the GGQ loopwith various amino acids in eL42 or the release factors havemodesteffects on cell growth and/or the activity of the release factors(Fig. 2B and Table S1). These results suggest comparable roles of theglutamine and central glycine residues of the GGQ loop in the eL42proteins and the release factors. By contrast, while mutations of thefirst glycine residue of the GGQmotif (Gly-51 in S. pombe) to alanineor arginine in eL42 have no effect on cell viability or growth (Fig. 2Aand B), they result in lethality when the respective substitutionsconcern the release factors (Table S1 and [20,31]).

3.3. Amino acid changes within or adjacent to the GGQ motif ofeL42 result in reduced activity of S. pombe 80S ribosomes

To address the question of the functional role of the GGQ min-idomain of eL42, and to establish the mechanism by which theprotein contributes to the activity of 80S ribosomes in the viableyeast cells, we compared the activities of the S. pombe wild-typeand mutants ribosomes in the poly(U)-dependent poly(Phe) syn-thesis. It should be mentioned that translation elongation in fungiwas previously shown to require a third protein, EF-3, in addition tofactors 1 and 2 [35]. This yeast-specific elongation factor stimulatesbinding of the ternary complex (aminoacyl-tRNA.GTP.EF-1a) to theribosomal A-site by facilitating the release of deacylated tRNA fromthe E-site. The latter reaction requires EF-3-dependent ATP hy-drolysis [35,36]. Therefore, measurement of the poly(U)-directedpoly(Phe) synthesis activity of the S. pombe wild-type and mutantribosomes in the conventional conditions that are used for thehuman 80S ribosomes [16] could not be achieved without thepresence of the elongation factor 3 (EF-3) from Saccharomycescerevisiae.

In order to determine the concentrations of S. pombe 80S ribo-somes that would ensure reliable activity measurements in steadystate conditions, we measured the activity of the wild-type andmutant ribosomes in the poly(U)-dependent poly(Phe) synthesis asa function of their concentration in the incubation mixtures. Asshown in Fig. 3A, the activities of the wild-type and mutant ribo-somes in poly(Phe) synthesis were found to be proportional to theirconcentration (up to 500 nM). Notably, the activities of the Q53Kand K55R mutant ribosomes were decreased by about 40% onaverage, as compared to those of the wild-type ribosome, whileactivities of the G51A, G51R and P56Q mutants remained un-changed (Fig. 3A). In Fig. 3B, the concentration of S. pombe wild-type or mutant 80S ribosomes is set at 0.4 mM where the kineticsof poly(Phe) synthesis correspond to those of steady state

conditions. In these conditions, the activities of all mutant ribo-somes were comparable with those measured in Fig. 3A, as ex-pected (Fig. 3B). Since the activity of ribosomes in protein synthesisis composed of the peptidyl transferase reaction followed by thetranslocation step, these results could suggest that Gln-53 and Lys-55 residues of the eL42 protein from yeast might be involved ineither of these steps on the ribosome. Finally, the peptidyl trans-ferase reaction with respect to puromycin [37] was comparablyreduced as that of the poly(U)-dependent poly(Phe) synthesis ac-tivity in the cases of the Q53K and K55R mutant ribosomes,whereas the activities of the G51A, G51R and P56Q mutants wereonly slightly affected. The fact that the poly(Phe) synthesis activityand the peptidyl transferase reaction were similarly affected in thewild-type andmutant ribosomes suggests that the 40% reduction inactivity of the Q53K and K55Rmutant ribosomes was caused by thenucleophilic attack step of peptidyl transfer. If so, binding to the A-site would not be restricted at all. We addressed this question bycomparing the enzymatic binding of [3H]Phe-tRNAPhe mediated byelongation factor-1a (EF-1a) to the A-site of poly(U)-programmedwild-type and mutant ribosomes. As shown in Fig. 4, [3H]Phe-tRNAPhe binds to the ribosomal A-site in the presence of EF-1a,whatever the nature (wild-type or mutant) of the eL42 proteincontained in the S. pombe ribosomes. These results would suggestthat occupation of the ribosomal A-site is unaffected in all mutantribosomes.

3.4. Cross-linking in situ of eL42 with tRNAox on S. pombe 80Sribosomes

Next, we have compared the yields of cross-linking of theribosome species to tRNAox. This reactive tRNA analogue was ob-tained by the periodate treatment of native tRNA, which specificallyoxidizes the 20,30-cis-diol function of the 30-terminal ribose of tRNA.It has been previously described as a powerful reagent for affinitylabeling of aa-tRNA synthetases [38e42]. More recently, tRNAoxwas found to be cross-linked with Lys-53 of eL42 in situ on human80S ribosomes [15,16]. This lysyl residue corresponds to Lys-55 ofeL42 fromyeast. Therefore, one can expect that tRNAox could cross-link with Lys-55 of eL42 in the yeast 80S ribosomes. Affinity la-beling of an aa-tRNA synthetase or a ribosomal protein by tRNAoxconsists in the formation of a reversible Schiff base between the20,30-aldehyde groups of tRNA and the ε-amino group of lysineresidues at the respective tRNA-CCA binding sites. The equilibriumfor Schiff base formation is displaced by reduction with sodiumcyanoborohydride [38]. Fig. 5A shows the analysis by 10% urea-polyacrylamide gel electrophoresis (urea-PAGE gel run as inRef. [15]) of the ribosomal proteins cross-linked to tRNAox at pH 7.5in the wild-type and mutant 80S S. pombe ribosomes. At this stage,it was verified by western blotting with specific antibodies againsteL42, that the protein cross-linked in majority with tRNAox in situon the wild-type S. pombe ribosomes (corresponding to the tRNA-eL42 band on Fig. 5A) is effectively eL42, as previously demon-strated for the human 80S ribosomes ([15,16] and results notshown). On Fig. 5A, the yields of cross-linked eL42, as estimated byscanning the gel with PhosphoImager were 63% for the wild-typeribosomes and 74%, 76%, 60%, 54% and 49% for the P56Q, K55R,Q53K, G51R and G51Amutant ones, respectively. It should be notedthat, as shown in Fig. 5A, other ribosomal proteins were also foundslightly labeled with tRNAox. Interestingly, the same cross-linkingpattern had been previously observed with the human 80S ribo-somes on urea-PAGE gels run in the same conditions [15], sug-gesting that the CCA-end of tRNAox binds similarly to eL42 on theS. pombe or the human 80S ribosomes. Unfortunately, in the presentstudy (Fig. 5A), as well as in previous reports [15,16], the rps labeledin minority with tRNAox contained in the weakly radioactive bands

Fig. 3. Effects of amino acid changes within or adjacent to the GGQ minidomain of eL42 on the activity of 80S ribosomes from S. pombe. (A), the poly(Phe) synthesis activitiesof poly(U)-programmed wild-type or mutant 80S ribosomes of Schizosaccharomyces pombe were measured as a function of the concentration of ribosome in the incubationmixtures. Values on the ordinate axis represent the amount (cpm) of [14C]phenylalanine incorporated into the poly(Phe) polypeptide in the presence of the wild-type or the mutantribosomes. The data were fitted with Mathematica to linear functions whose slopes were inscribed in the figure. The confidence intervals for the prediction of each slope were 95%.(B), kinetics of poly(Phe) synthesis by poly(U)-programmed wild-type or mutant 80S ribosomes. (C), the peptidyl transferase activity of the G51R, G51A, Q53K, K55R and P56Qmutant ribosomes was expressed as a percent of the activity of the wild-type ribosome considered as 100%. Each value for the peptidyl transferase activity was the mean value fromthree sets of experimental data.

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(Fig. 5A and [15]) were not in sufficient amount to permit proteinidentification from the urea-PAGE gels [15]. Therefore, eL42 can beconsidered as the only major rp labeled with tRNAox on theS. pombe or the human 80S ribosomes, consistent with a functionalrole for this protein at the tRNA-CCA binding site on the ribosomesof eukaryotic cells. Attempts to identify Lys-55 of S. pombe eL42 asthe amino acid residue cross-linked with tRNAox were not suc-cessful because of technical problems. In fact, we had previouslyreported that MALDI mass spectrometric identification of tRNAox-labeled peptides of human eL42 is very difficult due to the weak-ness of the signal of the cross-linked peptides and to mass de-viations between calculated and measured masses [16].Nevertheless, we have identified indirectly the Lys residue cross-linked with tRNAox by using the following approach: we havedesigned two recombinant S. pombe eL42 species named eL42-D(GGQTK) that is the eL42 protein whose GGQTK motif has beendeleted, and eL42-K55Q, a single K55Q mutant in which the criticalLys-55 residue was changed to Gln [27]. These eL42 variants wereincubated in the presence of tRNAox. As shown in Fig. 5B, tRNAoxfailed to cross-link with both the eL42-D(GGQTK) and the eL42-K55Q mutants lacking the Lys-55 residue. This result suggeststhat, as expected from previous affinity labeling studies, Lys-55 ofS. pombe eL42 is actually the tRNAox-labeled amino acid residue,similarly to Lys-53 of human eL42 [16].

3.5. pK of the Lys-55 residue of S. pombe eL42 cross-linked in situ totRNAox in the wild-type or mutant yeast 80S ribosomes

The question of the functional role of Lys-55 of eL42 in yeastribosomes was addressed by determining the pK value of thisresidue through the cross-linking reaction. To this purpose, wehave followed the cross-linking in situ with tRNAox of the eL42protein in yeast wild-type or mutant 80S ribosomes as a function ofthe pH of the incubation mixture. For example, Fig. 5C shows theincorporation of [32P]tRNAox into eL42 on the S. pombe wild-type80S ribosomes as a function of pH. On one hand, the eL42-tRNAox covalent complex has an apparent molecular weight of36,000 þ 1000 Da consistent with the formation of a covalentcomplex containing one polypeptide chain of endogenous S. pombeeL42 (about 12,000 Da) and one molecule of tRNAox (25,000 Da;calculated MW of the covalent eL42-tRNAox complex 37,000 Da)(Fig. 5C). Interestingly, the eL42 protein crosslinked with tRNAox insitu on the human 80S ribosomes had been previously shown topresent an apparent molecular weight of 36,000± 1000 Dacomprising one molecule of eL42 (molecular weight 12,000Da) andone molecule of tRNAox (25,000Da) [17]. On the other hand, theamount of [32P]tRNAox incorporated into eL42 on the wild-type orthe mutant S. pombe 80S ribosomes was shown to increase as afunction of increasing pH values (Fig. 5C). In total, estimation of the

Fig. 4. Enzymatic binding of [3H]Phe-tRNAPhe to the A-site of poly(U)-programmedS. pombe 80S ribosomes containing wild-type or mutant eL42 proteins. Comparisonof enzymatic binding of [3H]Phe-tRNAPhe to the A-site of poly(U)-programmed humanwild-type or mutant S. pombe 80S ribosomes. Values on the ordinate axis represent thepercent of bound [3H]Phe-tRNAPhe in relation to the total amount of 80S ribosomes inthe binding assays. Non-enzymatic or enzymatic binding in the absence or in thepresence of EF-1a (abscissa) are shown. For details see Experimental procedures. Eachvalue for the [3H]Phe-tRNAPhe binding to the A-site was obtained as the mean valuefrom two data sets of two separate experiments with each of the wild-type or mutantribosomes.

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cross-linking yields at pH 5.0, 6.0, 7.0, 7.5, 8.0 and 9.0 for the wild-type, and the P56Q, K55R, Q53K, G51R and G51Amutant ribosomesled to a value of 6.95± 0.05 for the side chain of Lys-55 (or Arg-55)of S. pombe eL42 (Fig. 5D). Noteworthy, it was verified that thepoly(U)-dependent poly(Phe) synthesis activity of the wild-type ormutant ribosomes was not significantly affected at pH values 5.0 to9.0.

To evaluate the influence of the neighboring ribosomal com-ponents on the pK of Lys-55 of eL42, we have followed the cross-linking reaction between recombinant S. pombe eL42 and tRNAoxas a function of the pH of the incubation mixture. Fig. 5E shows thevariation of the yield of the product of cross-linking as a function ofpH. The data from Fig. 5E were used to plot the cross-linking yieldversus increasing pH values, and a pK value of 6.9± 0.1 wasdeduced for the ε-amino group of Lys-55 (Fig. 5F). Similar pK valuesfor the ε-amino group of Lys-55 found with the S. pombe recom-binant eL42 and the ribosome-bound eL42 (see above) indicate thatother ribosomal components do not influence significantly thereactivity of this critical amino acid residue inside the S. pombe 80Sribosome.

4. Discussion

In this study, we performed the mutational analysis of theGFGGQTKP motif of the S. pombe eL42 protein with the goal ofdetermining the structural and functional roles of specific aminoacid residues in the GGQ minidomain common to the RFs andeukaryotic large subunit ribosomal proteins of the eL42 family. Allthe mutations operated in this motif of eL42 in viable S. pombe cellswere shown to lead to the impairment of activities of mutants ri-bosomes in poly(Phe) synthesis or in peptidyl transfer, as comparedwith those of the wild-type ribosome. In addition, a functional rolefor Lys-55 of eL42 in yeast ribosomes was revealed by estimatingthe pK value of this residue through the cross-linking of the protein

to P site-bound tRNA bearing oxidized 30-terminal ribose atdifferent pH. The data obtained bring new insights into the mo-lecular mechanism of peptide bond formation in eukaryotictranslation, highlighting the contribution of eL42 to the catalyticactivity of 80S ribosomes via the involvement of its GFGGQTKPmotif in peptidyl transfer to A-site-bound aa-tRNA, and/or in thehydrolysis of P site-bound peptidyl-tRNA.

4.1. The GGQ minidomain and neighboring regions of S. pombe eL42are critical for ribosomal function in vivo and in vitro

S. pombe cells expressing mutant rpl42 genes for G51R, G51A, orQ53K could be successfully isolated using a conventional genereplacement method, while cells expressing mutant rpl42 genewith six amino acids deletion (51-GGQTKP-56) or with the G49A,G52R, K55Q, or K55E substitutions could not. Moreover, whenmutant eL42 proteins from an ectopic plasmid were expressed inwild-type yeast cells, those expressing D51-56, G49R, G52R, orG52A mutants showed severe growth defects, while a moderategrowth defect was seen for the G49A, G51R, G51A, Q53K, or K55Qmutants. These genetical data suggest that some of the amino acidresidues of the GFGGQTKP motif are critical to the function ofS. pombe eL42. It is interesting to note that, as revealed by com-parison of Fig. 2A and Table S1, the data obtained in this studyresemble those reported by several research groups on theeukaryotic and prokaryotic release factors [20,22,31e33]. In fact, ithas been previously reported that the GGQ minidomain of thehuman translation termination factor eRF1 contains a 181Gly-Arg-Gly-Gly184 tetrapeptide unique in all available eRF1 amino acidsequences, the invariant form of which is 181Gly-X-X-Gly184 [22].This invariant Gly-X-X-Gly form is found conserved in theeukaryotic (eRF1) and the prokaryotic (RF1, RF2) release factors[20,22,31e33,43], as well as in the eL42 proteins from all eukaryotic80S ribosomes (49Gly-X-X-Gly52 in S. Pombe). Besides, the proteinsthat exhibit a peptidyl-tRNA hydrolase activity also comprise thisinvariant tetrapeptide (Fig. 1 and [44e48]). Therefore, failure toisolate S. Pombe cells producing the G49A, G49R, G52A or G52Rmutants is consistent with the fact that, with the exception of a fewarchaeabacterial eL42, these glycyl residues are strictly conservedin all the proteins of Fig. 1. By using the same in vivo genetical ap-proaches as those reported here, Song et al. [31], have demon-strated that substitution of any amino acid residue in the GGQtripeptide of the yeast eRF1 is lethal for yeast cells (see Table S1).Another example concerns the effects caused by extensive muta-genesis of conserved amino acid residues in the GGQ domain ofhuman eRF1 and of E. coli RF1. It turned out that mutations of theGGQ glycines (especially the second one, Gly-184 in human eRF1)lead to dramatic reductions in the rate constant for peptide release[20e23,32e34], suggesting a structural role for these residues. Inall these studies, the most popular interpretation of the observedeffects is that replacement of these glycines (especially the secondone) by Ala would prevent the GGQ loop from adopting the flexiblestructure that enables the factors to reach and activate the PTC.Similarly to the aforementioned data, our genetical data stronglysuggest that the effects of the replacements of the glycines in the49GFGG52 motif of S. pombe eL42 on cell growth likely reflect thatthese glycines play a structural role essential to the function of theribosome, and might contribute to particular step(s) in the trans-lation process.

4.2. Amino acid changes within or adjacent to the GGQ motif ofeL42 result in reduced activity of S. pombe 80S ribosomes

The poly(Phe) synthesis activity of the wild type and mutantribosomes from S. pombe was found to be proportional to their

Fig. 5. Cross-linking with tRNAox of S. pombe ribosomes containing the wild-type or mutant eL42 species, or of recombinant wild-type or mutant rps eL42. (A), S. pomberibosomes containing the wild-type or the P56Q, K55R, Q53K, G51R and G51A mutant species were incubated with [32P]tRNAAspox at pH 7.5, and the ribosomal proteins cross-linkedto tRNAox were analyzed by 10% urea-polyacrylamide gel electrophoresis (urea-PAGE gel run as in Ref. [15]). The gels were subjected to autoradiography as in Ref. [15]. The yields ofcross-linked eL42, as estimated by scanning the gel with PhosphoImager were 63%, 74%, 76%, 60%, 54% and 49%, for the wild-type, the P56Q, the K55R, the Q53K, the G51R and theG51A mutant ribosomes, respectively. Since six-lane gels were used in this experiment, the control ([32P]tRNAAspox alone in the absence of ribosomes) (lane tRNA) was taken from aseparate gel run in parallel in the same conditions. This control showed only the lower radioactive band on the gel (lane tRNA). (B), cross-linking of the recombinant S. pombe eL42or the eL42-D(GGQTK) and eL42-K55Q mutants with [32P]tRNAox. The recombinant wild-type S. pombe eL42 (lane 1) or the eL42-D(GGQTK) mutant (lane 2) as well as the singleK55Q (eL42-K55Q) mutant (lane 3) lacking the Lys-55 residue were incubated with tRNAox and analyzed by SDS-PAGE as described under Materials and Methods. The unreactedtRNAox and the eL42-tRNAox covalent complex are shown. The control ([32P]tRNAAspox alone) showed only the lower radioactive band (data not shown). (C), cross-linking of eL42with tRNAox in situ on wild-type or mutant S. pombe 80S ribosomes as a function of the pH of the incubation mixture. Wild-type S. pombe 80S ribosomes were incubated with [32P]tRNAAspox at pH 5.0, 6.0, 7.0, 7.5, 8.0 and 9.0, and the samples were subjected to SDS-PAGE and the resulting gels to autoradiography as in Ref. [15]. The cross-linking yields weredetermined as described earlier in Ref. [17]. (D), plot of the molar fraction of the cross-linked eL42-tRNAox complex as a function of the pH of the incubation mixture. The cross-linking yields at pH 5.0, 6.0, 7.0, 7.5, 8.0 and 9.0 for the wild-type and the Q53K, K55R and P56Q mutant ribosomes were determined as in (C). The data were fitted with Mathematicato the function inscribed in the figure, and a pK value of 6.95 ± 0.05 was deduced. The confidence intervals for the prediction of a single value were 95%. The curves of the G51R andG51A mutant ribosomes were superimposable on those of the Q53K, K55R and P56Q mutant ribosomes (data not shown). (E), cross-linking of recombinant S. pombe rp eL42 with[32P]tRNAox as a function of the pH of the incubation mixture. The cross-linked recombinant S. pombe rp eL42 was analyzed by SDS-PAGE on 10% polyacrylamide gels. The tRNA-eL42 covalent complex formed as a function of pH is shown. The control ([32P]tRNAAspox alone) run on a separate gel showed only the lower radioactive band (data not shown). (F),plot of the molar fraction of the cross-linked eL42-[32P]tRNAox complex as a function of the pH of the incubation mixture. The cross-linking yields with [32P]tRNAox at pH 5.0, 6.0,7.0, 7.5, 8.0 and 9.0 for the recombinant S. pombe eL42 were determined from (E). Two data sets from two separate experiments as in (E) were used to obtain this graph. The datawere fitted with Mathematica to the function inscribed in the figure with the following features: the dashed lines defines the 95% confidence intervals for the prediction of a singlevalue, whereas the continuous lines and the gray-shaded region define the 95% confidence intervals for the prediction of the average curve with pK¼ 6.9± 0.1.

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concentration in the incubation mixtures (up to 500 nM), on onehand, and to the incubation time (up to 40min), on the other hand.These results argue that the activity measurements were per-formed in the steady state conditions where the activity is specificfor the ribosome species under consideration. With these twocomplementary assays, the poly(Phe) synthesis activities of theQ53K and K55Rmutant ribosomes were found decreased by 40% onaverage, as compared to the activity of the wild-type ribosome,while those of the G51A, G51R and P56Q mutant ribosomesremained unchanged. In the case of the peptidyl transferase reac-tionwith respect to puromycin, the activities of the Q53K and K55Rmutant ribosomes were comparably decreased by 40% as inpoly(Phe) synthesis suggesting that the Q53 and K55 residuesmight be directly involved in the peptidyl transferase activity. In thecase of the K55R mutation, its effect on the peptidyl transferaseactivity likely reflects that the side chain of Arg is less nucleophilicthan that of Lys and, therefore, the nucleophilic attack at the step ofpeptidyl transfer would be less efficient. Consistent with this hy-pothesis, a few archaebacteria contain an Arg residue at the posi-tion of Lys-55 in the amino sequence of their eL42 protein (Fig. 1and [15]), assuming that the only requirement of the reactioncatalyzed or facilitated by eL42 is the nucleophilic character of the

critical amino acid residue at this position. Given that glutamineand glutamic acid are polar amino acid residues roughly compa-rable in size with lysine and containing a neutral or a negativelycharged side chain, respectively, it becomes clear why S. pombecells expressing mutant rpl42 genes corresponding to the K55Q orK55E substitutions are nonviable because of the presence ofnonfunctional ribosomes in these cells. As for the Q53K mutation,the effect of this substitution on the peptidyl transferase activity ofthe yeast 80S ribosome is reminiscent of the effect of the replace-ment of Gln-185 on the peptidyl-tRNA hydrolase activity of thehuman translation termination factor eRF1 (Table S1 and [22]).Contrasting with the poly(Phe) synthesis activity, the peptidyltransfer reaction of the G51A, G51R and P56Q mutant ribosomeswas slightly reduced, suggesting the Gly-51 and Pro-56 residuesmight participate in some way to the peptidyl transfer step in thecourse of the elongation of translation.

4.3. Abnormally low pK for the side chain of Lys-55 (or Arg-55)cross-linked in situ to tRNAox in the yeast 80S wild-type or mutantribosomes

The dialdehyde derivative of tRNA (tRNAox) introduces a simple

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way to affinity label the CCA-binding site of tRNA on ribosomalproteins. Moreover, the covalent reaction can be monitored indifferent conditions such as the variation of the pH of the incuba-tion mixtures of cross-linking that enables deduction of the pK ofthe amino acid side chains through the covalent reaction.

In the present report, cross-linking in situ to tRNAox of the eL42protein in the yeast 80S ribosomes as a function of the pH of theincubation mixture indicated a pK value of 6.95± 0.05 for the sidechain of Lys-55 (or Arg-55) of the wild-type ribosome and of theP56Q, K55R, Q53K, G51R and G51A mutant ones. It should berecalled that in a previous study, the cross-linking in situ to tRNAoxof the eL42 protein in the human 80S ribosomes as a function of thepH was performed and a pK value of 6.9± 0.1 was deduced for thecross-linked Lys-53 residue [17]. It is generally accepted thatcatalysis almost exclusively depends on the ionization of catalyticgroups, and that the function of proteins and enzymes is frequentlyassociated with an amino acid side chain having an unusual pK.Therefore, the fact that a pK value of 6.9e6.95 (instead of 10.5) wasmeasured for the ε-amino group of Lys-55 in yeast eL42 (or Lys-53in the human eL42) suggests that, in both eukaryotic 80S ribo-somes, these lysyl residues most probably play a functional role atthe PTC. A pK value of 6.95 was unexpected for the Arg-55 residue(K55R mutant ribosome) because the pK value of the side chain ofArg (12.5) is usually higher than that of Lys (10.5). In accordancewith this observation, we had previously demonstrated that theside chain of Arg is less reactive than that of Lys through Schiff baseformation with tRNAox [42]. Expectedly, in the present report, theactivity of the K55R mutant ribosome was decreased by 40%, ascompared to that of the wild-type ribosome, suggesting that thenucleophilic attack step of peptidyl transfer would be less efficientwith Arg. However, as shown in Fig. 5A, the yield of cross-linking ofArg-55 (K55R mutant ribosome cross-linked by 76%) is higher thanthat of the wild-type ribosome containing the critical Lys-55 res-idue (cross-linked by 63%), suggesting that this Arg-55 residuemight be at least as reactive as Lys-55 through the cross-linkingreaction. A possible explanation for the fact that the K55R mutanthas a pH profile similar to that of the wild-type eL42 despite thedifference of 2 units between the normal pK values of Lys and Arg isthat, actually, the in vivo naturally occurring peptidyl transfer re-action and the in vitro cross-linking reaction via a Schiff base for-mation are not of the same nature and do not rely on the samechemical mechanism. It is interesting to note that, as discussedabove, a few archaebacteria contain an Arg residue at the positionof Lys-55 in the amino acid sequence of their eL42 protein (Fig. 1and [15]), suggesting that Arg may represent an alternative nucle-ophile in the GGQ minidomain of the archaeal eL42 proteins. Inconclusion, taking into account the latter observation and thedemonstration that S. pombe cells expressing mutant rpl42 genescorresponding to the K55Q or K55E substitutions are nonviable, it ismost likely that, as discussed above, the only requirement of thereaction catalyzed or facilitated by rp eL42 is the positive charge ofthe side chain of the amino acid residue at position 55.

4.4. Binding and correct positioning of the incoming aa-tRNA at theA-site of S. pombe 80S ribosomes

The pK value of 6.9e6.95 assigned to the ε-amino group of Lys-55 (or Lys-53) of eL42 in human and in the yeast, indicates that theprotonated, positively charged εNH3

þ side chain of Lys-55 (or Lys-53) can be estimated to represent about 50% of this primaryamine group at neutral pH. Therefore, at this stage, one can antic-ipate that the role of the Lys-55 side chain of S. pombe eL42 mightconsist in the binding and the correct positioning of the incomingaa-tRNA at the ribosomal A-site or the catalysis of peptide bondformation, or both. Thus, at neutral pH, on one hand, the positively

charged εNH3þ group of Lys-55 is susceptible to establish ionic

bonds with phosphate groups of the polyanionic tRNA molecule,while, on the other hand, the remaining 50% of unprotonated εNH2form would be capable of performing catalysis of peptide bondformation. Since all the mutant ribosomes purified from viableS. pombe cells contained mutant eL42 proteins (G51A, G51R, Q53K,K55R and P56Q) with a Lys or Arg residue at position 55, one canexplain why the binding of Phe-tRNAPhe to the ribosomal A-site inthe presence of EF-1a is unaffected whatever the nature (wild-typeor mutant) of the eL42 proteins (Fig. 4).

4.5. Possible role of the Lys-55 side chain of S. pombe eL42 in thecatalysis of peptide bond formation

Regarding the catalytic activity, the presence of Lys-55 in theG51A, G51R and P56Q mutant ribosomes agrees well with theobservation that the poly(U)-dependent poly(Phe) synthesis ac-tivities of these mutant ribosomes remained unchanged (Fig. 3A).In addition, as discussed above, the fact that the activity of the K55Rmutant ribosome was decreased by 40%, as compared with that ofthe wild-type ribosome, is likely to reflect that the side chain of Argis less nucleophilic than that of Lys, and that the nucleophilic attackstep would be less efficient (Fig. 3A). The decrease of activity of theQ53K mutant ribosome will be discussed in another Section. Alto-gether, our data are consistent with the historical observation byseveral research groups that formation of a peptide bond dependson two ionizable groups, one with a pK value of 6.9, and the otherwith a pK value of 7.5 [49e51]. The former pK value was thought tobe associated with the a-NH2 group of puromycin used in the ex-periments, while the latter was attributed to a titratable group inthe active site of the ribosome, but this group had not been iden-tified so far. Remarkably, as discussed above, the ε-amino group ofLys-55 (or Lys-53) of eL42 in human and in the yeast was shown toexhibit a near neutral pK value of 6.9e6.95 (instead of 10.5), whileno pK value had ever been assigned to any chemical groupbelonging to the ribosome. Therefore, it is attractive to propose thatthe side chain of Lys-55 of the yeast eL42 (Lys-53 in human eL42)represents the single ionizing ribosomal group with a near neutralpK, which might be involved in catalysis.

4.6. Proposed overall mechanism for peptidyl transfer duringpoly(Phe) synthesis by S. pombe 80S ribosomes

Altogether, the data reported here support the participation ofthe lysyl residue 55 of eL42 to the activity of the yeast 80S ribo-somes. The hypothetical mechanism for peptidyl transfer that wepropose for S. pombe 80S ribosomes is shown in Fig. 6A. The startingpoint in the elongation of translation is the binding and the correctpositioning of the incoming aminoacyl-tRNA at the ribosomal A-site by rp eL42 by means of electrostatic interactions between thepositively charged side chain of Lys-55 and phosphate groups of theCCA-arm of the tRNA molecule (Fig. 6A). This is followed by thedeprotonation of the a-NH3

þ group of the aa-tRNA positioned at theA-site by the unprotonated εNH2 group of the catalytic Lys-55 sidechain to generate the nucleophilic a-NH2 group capable of attack-ing the ester bond of peptidyl-tRNA in the P-site (Fig. 6A). Thesetwo events constitute the first step of the mechanism (Fig. 6A, Step1). In the second step, the nucleophilic attack of the a-NH2 group onthe electrophilic carbonyl group leads to peptide bond formationand to subsequent deacylation of the P-site tRNA, as well as toprotonation of its 30-end (Fig. 6A, Step 2). Finally, in the third step,the catalytic ε-NH3

þ group of Lys-55 donates a proton to a watermolecule, in order to restore the nucleophilic ε-NH2 group capableof abstracting a proton from the a-NH3

þ group of the incoming aa-tRNA to initiate the next elongation cycle (Fig. 6A, Step 3). This

Fig. 6. Schematic of the proposed model for the mechanisms of peptidyl transfer and release factor-mediated peptidyl-tRNA hydrolysis involving eL42 of human 80S ribosome. (A), the overall hypothetical mechanism ofpeptide bond formation by S. pombe 80S ribosomes. (B), stepwise peptidyl transfer reaction consisting of the nucleophilic attack of the a-amino group of A-site bound aminoacyl-tRNA (aminolysis) on the P-site substrate peptidyl-tRNA.(C), hydrolysis of peptidyl-tRNA ester bond in P-site by a catalytic water molecule coordinated by Gln-185 of the eRF1 GGQ tripeptide in A-site at the translation termination. In both cases, the human eL42 protein would act as thecatalytic base that assists peptidyl transfer and release factor-mediated peptidyl-tRNA hydrolysis. This figure was adapted from Song et al., [31].

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situation is comparable with that of Lys-65 of E. coli rp bL12 thatwas previously shown to be critical for the poly(U)-dependentpoly(Phe) synthesis activity of E. coli 70S ribosomes [52]. In fact,we have recently demonstrated that the Lys-65 side chain partici-pates to the binding of the incoming aa-tRNA to the ribosomal A-site through ionic bonds with phosphate groups and, at the sametime, performs the nucleophilic attack of its a-amino group on thecarbonyl carbone of the peptidyl-tRNA positioned at the P-site ofE. coli 70S ribosomes [53].

4.7. Fine tuning of the nucleophilic attack step

The basic overall mechanism for peptidyl transfer common tothe eukaryal and eubacterial [53] ribosomes described in the pre-sent report is compatible with the widely accepted idea that themajor steps (tRNA binding to the ribosome, decoding, peptide bondformation, etc) involved in the elongation step of translation shouldbe the same whatever the origin of the ribosome. However, it ismost probable that pronounced differences exist between theeubacteria and the eukaryotes that might be related to a morecomplex structure of the eukaryal ribosomes. Such a differencemight for example be accounted for by the presence of a glutamineresidue in proximity to the catalytic Lys-55 residue of S. pombeeL42. In fact, eukaryotic large subunit ribosomal proteins of theeL42 family exhibit the universally conserved GXGGQ motif typicalfor release factors (RFs) responsible for triggering peptidyl-tRNAhydrolysis during translation termination (Fig. 1 and[20,22,31,32,43]). Moreover, it is generally accepted that the PTCparticipates in the catalysis of two chemical reactions. The first isthe formation of peptide bonds referred to as aminolysis i.e., thenucleophilic attack of the a-amino group of the A-site bound aa-tRNA on the P-site substrate peptidyl-tRNA. The second is therelease factor-mediated hydrolysis of peptidyl-tRNA upon trans-lation termination involving the nucleophilic attack of a watermolecule (in the A-site) on the peptidyl-tRNA. Since themechanismfor peptide bond formation is strikingly similar to that of peptiderelease, it is not unlikely that both reactions could use the sametype of catalytic residue brought by eL42 in one case, and by eRF1 inthe other. Therefore, one can reasonably imagine that these ribo-somal proteins participate to catalysis in the sameway as the RFs dowhen they use a water molecule for the nucleophilic attack on thecarbonyl carbone of P-site bound peptidyl-tRNA, at the end oftranslation (Fig. 6B and C). It should be noted that our model isquite similar to that of Song et al. [31], on transesterification reac-tion where, as in our hypothetical model proposed in the presentreport, a chemical group that would supposedly represent the PTCis needed (Fig. 6B).

4.8. Rationale for a functional role of the GGQTKP motif of eL42

In addition to the genetical and biochemical data reported here,the following data also demonstrate that the GGQ minidomaincommon to the RFs and the eL42 proteins plays a functional role: (i)first, we have recently demonstrated that the CCA-end of a tRNA inthe P/P state interacts in majority with a lysyl residue (Lys-197) inthe neighborhood of the GGQmotif of eRF1 bound to an A-site stopcodon [54]. This result suggests that the CCA-arm of P-tRNA bindsto the GGQ minidomain of eRF1, in accordance with crystallo-graphic data [31]. Moreover, this interaction is of functionalimportance, since it was previously shown to result in the hydro-lysis of P-site bound peptidyl-tRNA, at the termination of trans-lation [20,22,31,32,43]; (ii) second, in another study cited above[17], we have demonstrated that the CCA-end of a P-tRNA interactswith both GGQ minidomains of eL42 and of an A-site boundtranslation termination factor eRF1. This result suggests that,

similarly to the GGQ minidomain of A-site bound eRF1, the GGQminidomain of eL42 is likely to play a functional role, as proposed inFig. 6. The latter interpretation agrees well with the observationthat the effects of mutational analyses of the GGQ minidomain ofeRF1 are quite similar with those of eL42 (Fig. 2A and Table S1); (iii)third, electron cryomicroscopy studies on Saccharomyces cerevisiae80S ribosomes have indicated that, in the 3-D structure at 6 Åresolution of these ribosomes in complexwith two tRNAmolecules,one tRNA molecule was found at the E-site with the CCA-arminserted into the GGQ loop of eL42, while the other was bound atthe interface of the P- and E-sites [55]. This result is similar to theone obtained by the Yusupov group [60] with X-ray crystallo-graphic studies, and confirms at the same time a critical role for theGGQ loop of the eukaryal or the archaeal eL42.

4.9. Lys-55 of the GGQTKP motif of eL42 is a target for small-molecule inhibitors of protein biosynthesis by the yeast ribosomes

Another argument which is in favor of a critical role for theGGQTKPmotif of eL42 in relationwith our hypothetical mechanismfor peptidyl transfer is that Lys-55 of this motif is a target for small-molecule inhibitors of ribosomal protein biosynthesis. For example,cycloheximide, a strong inhibitor of translation in eukaryotic cellshad been shown to bind to Lys-55 of the GGQTKP motif of eL42 inS. cerevisiae 80S ribosomes [56]. In addition, several yeast strainsare known to exhibit resistance mutations. One of these mutations,P56Q of eL42, renders them resistant to cycloheximide [28,56e58].The fact that cycloheximide inhibits protein biosynthesis by bind-ing to Lys-55 of eL42, while the mutation P56Q renders some yeaststrains resistant to cycloheximide, indicates that these two adjacentamino acid residues (Lys-55 and Pro-56) are involved in the bindingand in the inhibitory effect of cycloheximide on the activity of theribosome. A neighboring ribosomal protein, uL15 (formerly L28 inyeast or L27a in mammals) has been also implicated in cyclohexi-mide resistance. It is interesting to note that uL15 contains a36GGQ38 motif in the peptide 30GGRGMAGGQHHHR42, and aQ38E transitionwas responsible for the resistance [59]. Fig. 7 showsthat both GGQ motifs of eL42 and uL15 (taken from the best reso-lution, 2.8 Å, PDB file 4u4r) are involved in the binding of cyclo-heximide (4u3u) [56]. Moreover, in the crystallographic structure ofS. cerevisiae 80S ribosomes or of the 50S subunit of Haloarculamarismortui, most of the translation inhibitors were shown totarget the eL42 protein and to make contact with the lysyl residue(Lys-55 in S. cerevisiae and Lys-51 in Hma) of the extension loopcontaining the GGQTKP peptide (GNDGKF in Hma) [56,60e62]. Thefact that Lys-55 of the GGQTKP motif of the yeast eL42 (Lys-53 inhuman or Lys-51 of GNDGKF in Hma) is a target for small-moleculeinhibitors of protein biosynthesis argues for a critical role for thislysyl residue at the PTC of the ribosomes, in agreement with ourmodel shown in Fig. 6. Finally, Fig. 7 shows the yeast eL42, eL15 anduL15 proteins (PDB file 4u4r) bound to the trinucleotide CCA (4u3n)[56]. We observe that (i) Tyr-43 in the helical region of eL42 isbound to the C75 or C74 nucleotide of the tRNA CCA-arm, and (ii)Phe-50 in the tight loop region 49GFGG52 of eL42 is bound to the84PTNQGVNE91 of eL15. Thus, the PDB data show that these in-teractions bring the CCA-arm close to the Gln-53 and Lys-55 resi-dues, in accordance with the proposition that catalysis could beassisted by the GGQTK peptide through the Gln-53 and Lys-55residues.

5. Conclusion

With the design of genetic experiments stemming from previ-ous affinity labeling studies, we found that the region of GGQ motifof eL42might play a role crucial for polypeptide chain elongation in

Fig. 7. Location of the eL42, eL15 and uL15 proteins in the 3-D structure of the 80S ribosome from Saccharomyces cerevisiae. The large subunit ribosomal proteins eL42, eL15and uL15 (PDB accession code 4u4r), bound to the trinucleotide CCA (PDB accession code 4u3n) and to cycloheximide (PDB accession code 4u3u), as reported in Ref. [56], are shown.

C. Hountondji et al. / Biochimie 158 (2019) 20e3332

translating ribosomes from S. pombe cells. This led us to propose anhypothetical mechanism where, at neutral pH, on one hand, thepositively charged εNH3

þ group of Lys-55 establishes ionic bondswith phosphate groups of the polyanionic tRNAmolecule, while, onthe other hand, the remaining 50% of unprotonated εNH2 performsthe catalysis of peptide bond formation. According to the previousdemonstration by our group that the CCA-end of a P-tRNA interactswith both GGQ minidomains of eL42 and of an A-site boundtranslation termination factor eRF1, we propose closely relatedmechanisms for peptidyl transfer at the elongation step and for therelease factor-mediated peptidyl-tRNA hydrolysis at the termina-tion of translation. The catalytic Lys-55 side chain of eL42 is sup-posed to withdraw a proton from the a-NH3

þ group of A-site boundaa-tRNA or from the hydrolytic water molecule prior to theirnucleophilic attack on the carbonyl carbone of P-site bound pep-tidyl-tRNA.

Competing interests

No competing interests are declared.

Authors' contributions

C.H., J.N., G.K. and S.B. designed and planned experiments; J.B.C.,K.B., M.T., M.S., B.A., C.H. and S.B. carried out experiments; J.N., M.T.and M.S. prepared DNA constructs encoding for mutant S. pomberibosomes; J.B.C. prepared DNA constructs encoding for mutantrecombinant S. pombe rp eL42; J.A.H.C. and C.H. interpreted thekinetics and exploited protein data bank and extracted a figurefrom it; all authors were involved in analysis and discussions of theresults obtained in this study; C.H., J.N., G.K. and J.A.H.C. wrote thepaper.

Funding

This work was supported by MEXT KAKENHI (grant # 17054045to JN).

Acknowledgements

We are grateful to Drs. Terri Kinzy and Maria Mateyak for the

kind gift of an E. coli strain expressing 6xHis-tagged eEF3. We thankDr. Atsuko Shirai for her initial effort tomake S. pombe rpl42mutantstrains. We gratefully thank Drs. Eric Guittet, G�erard Keith and Jo€elPothier for their constant interest for this work, and for fruitfuldiscussions.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.biochi.2018.12.005.

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