Interactions of elongation factor EF-P with the Escherichia coli ribosome Hiroyuki Aoki 1 , John Xu 1 , Andrew Emili 2 , John G. Chosay 3 , Ashkan Golshani 4 and M. Clelia Ganoza 1 1 University of Toronto, C.H. Best Institute, Canada 2 Terrance Donnelly Centre for Cellular and Biomolecular Research (Donnelly CCBR), Toronto, Canada 3 Pfizer Pharmaceuticals, Ann Arbor, MI, USA 4 Department of Biology, Carleton University, Ottawa, Canada In all cells, ribosomes are inert in the absence of several proteins that promote each facet of protein synthesis. In eubacteria, initiation factors (IF-1, IF-2, IF-3) bind to the 30S subunit, whereas elongation factors (EF-Tu and EF-G) and termination factors (RF-1, RF-2 and RF-3) bind to 70S ribosomes. Reconstitution of synthesis using all of these homoge- neous proteins revealed that they are insufficient for synthesis directed by a native mRNA and that several other proteins are required [1–6]. One of these pro- teins, EF-P, stimulates peptide bond synthesis by 70S ribosomes [1,2,6–8]. The gene encoding EF-P, efp, occurs at a unique site at 94.3 min on the Esc- herichia coli chromosome [9]. Interruption of the efp gene is lethal to the cell and results in an abrupt cessation of protein synthesis, specifically in an impairment of peptide-bond formation [10]. Cells deleted for efp grow only in the presence of the efp gene in trans. Thus, the efp gene is essential for cell growth and viability [10,11]. Reconstitution experiments and the effect of various antibiotics suggest that EF-P binds to a site on ribo- somes that differs from the binding site of most other translation factors. Thus, EF-P requires ribosomal pro- tein L16 for its action, but not L7 ⁄ L12, L6 or L11 [12]. All of the G proteins, IF-2, EF-Tu, EF-G and RF-3 act with L7 ⁄ L12 in the sarcin-ricin loop to trig- ger GTP hydrolysis, and L6 is required for the action of the release factors [6]. Several antibiotics perturb the action of EF-P. Notably, streptomycin, which impairs translational fidelity, is a strong inhibitor of the EF-P reaction [12]. However, other antibiotics that alter the fidelity of decoding such as neomycin and kasugamycin, have no effect on the EF-P-mediated Keywords A-site; eIF5A; elongation factor EF-P; ribosomes; translocation Correspondence H. Aoki, University of Toronto, C.H. Best Institute, 112 College Street, Toronto, Ontario M5G 1L6, Canada Fax: +1 416 978 8528 Tel: +1 416 978 8918 E-mail: hiro.dr.aoki@utoronto.ca (Received 1 August 2007, revised 3 Decem- ber 2007, accepted 10 December 2007) doi:10.1111/j.1742-4658.2007.06228.x EF-P (eubacterial elongation factor P) is a highly conserved protein essen- tial for protein synthesis. We report that EF-P protects 16S rRNA near the G526 streptomycin and the S12 and mRNA binding sites (30S T-site). EF-P also protects domain V of the 23S rRNA proximal to the A-site (50S T-site) and more strongly the A-site of 70S ribosomes. We suggest that EF-P: (a) may play a role in translational fidelity and (b) prevents entry of fMet–tRNA into the A-site enabling it to bind to the 50S P-site. We also report that EF-P promotes a ribosome-dependent accommodation of fMet–tRNA into the 70S P-site. Abbreviations CMCT, 1-cyclohexyl-3-(2-morpholinoethyl)carbodi-imide metho-p-toluenesulfonate; DMS, dimethyl sulfate; EF-P, eubacterial elongation factor P; IF, initiation factor; Ke, kethoxal; RF, termination factor. FEBS Journal 275 (2008) 671–681 ª 2008 The Authors Journal compilation ª 2008 FEBS 671 synthesis of peptide bonds [12]. Also, translocation inhibitors have little or no affect on the EF-P reaction [12]. EF-P enhances the inhibition of peptide-bond for- mation by chloramphenicol and lincomycin [12]. These results imply that EF-P acts near the peptidyltransfer- ase center of the ribosome. EF-P is required for synthesis with native mRNA templates and for synthesis of poly(Phe) primed by N-acetyl-Phe-tRNA as the initiator [1,2,12]. The EF-P protein is essential for the synthesis of certain fMet-ini- tiated dipeptides [8,12]. Thus, the action of EF-P may be similar to that of initiation factor eIF-5A in eukary- otes, which preferentially promotes synthesis of the first peptide bond in the protein sequence [13–15]. This is further substantiated by the fact that EF-P harbors 84 and 64% sequence similarity with aIF5-A from archaebacteria and eIF-5A from eukaryotes, respect- ively [16–18]. Remarkably, the crystal structure of EF-P is an L, or tRNA-like shape, characteristic of many proteins that actuate translation [16–18]. The function of EF-P and eIF-5A are currently unknown. It has been proposed that these proteins stimulate the synthesis of certain proteins in the cell [19]. In mammalian cells, including in humans, the genes encoding eIF-5A and its isoform, eIF-5A2, are oncogenes [19]. Despite these observations, the specific mode of action of these proteins in translation remains enigmatic. Here, we examine interactions between EF-P and 70S ribosomes using several rRNA structure-specific chemical probes [20,21]. The nature of the interactions between specific rRNA domains and EF-P suggests a plausible mode of action for this ubiquitous protein. Results Three approaches were undertaken to study the posi- tion of the EF-P protein on the Escherichia coli 70S ribosome and its subunits. Binding of labeled EF-P to ribosomes To study the binding site of EF-P on ribosomes, we first ascertained that ribosomes were free of EF-P. This was accomplished by washing the ribosomes in 0.5 m NH 4 C1 buffer and isolating the 70S peak after two successive sucrose-density gradient centrifugation steps as described in Experimental procedures [22]. Western blotting of the purified 70S preparations revealed that the ribosomes were free of EF-P (data not shown). A specific anti-EF-P mouse mAb was used to detect the protein. The purity of the EF-P was first assessed by isoelec- tric focusing in the first dimension followed by SDS electrophoresis as described previously [23] and by subsequent immunoblotting with specific anti-(EF-P) mAbs. A single band was observed on Coomasie Bril- liant Blue-stained gels and on the corresponding im- munoblots of the EF-P protein (Fig. 1A). Thus, the EF-P protein appears to be homogeneous by these cri- teria. This was verified by MS analysis of the tryptic fragments of the protein (see Experimental proce- dures). From the MS analysis of EF-P it was found that the residue blocking Lys34 conforms to spermi- dine within ± 1 Da (Table 1). No other known com- pound was found to more closely fit this mass. The N-terminus of the purified EF-P protein was labeled by formylation with [ 14 C]formate and the protein was bound to partly dissociated 70S ribo- somes containing 30S and 50S subunits, as described in Experimental procedures. The ribosomeÆ[ 14 C]EF-P complex was sedimented on sucrose-density gradients and the radioactivity of the different fractions was mea- sured. EF-P was found associated with 70S ribosomes and 30S and 50S subunits. Table 2 shows the amount of EF-P that binds to each subunit and to 70S ribo- somes. The labeled protein binds in an  1 : 1 molar ratio to 70S ribosomes and to 30S and 50S subunits. Identification of EF-P by immunoprecipitation of ribosomes and their subunits To examine whether EF-P occurs bound to native polyribosomes, a mouse mAb specific to EF-P was used to detect the protein. As shown in Fig. 1B, the EF-P protein can be detected on 70S,30S and 50S particles and on polyribosomes. Fractions from the sucrose-density gradients were collected and sub- jected to isoelectric focusing in the first dimension followed by 1D SDS electrophoresis and western blotting. As shown in Fig. 1B, EF-P is bound to the 30S and 50S subunits, to 70S ribosomes and to poly- ribosomes. The density of the immunoblots was determined, as was the total area of each peak in the ribosome profile. The percentage of bound EF-P protein in each peak shows that EF-P is distributed equally between 30S and 50S subunits and on 70S ribosomes. Approximately 17% of the protein was found on 70S ribosomes and 7% occurred bound to the 30S and 50S subunits, respectively. The reminder of the protein was recovered in the polyribosome fraction. The bound EF-P fraction decreases as a function of the number of ribosomes in the polyribosome fractions (Fig. 1C). This suggests that EF-P acts in an initial stage of synthesis. Ribosome interactions with elongation factor EF-P H. Aoki et al. 672 FEBS Journal 275 (2008) 671–681 ª 2008 The Authors Journal compilation ª 2008 FEBS EF-P binding site on the ribosome To further examine the interactions of EF-P, ribo- somes were treated with various base-specific reagents that react with unpaired bases at the ring nitrogen of each exposed rRNA base. The sequence of the probes used spanned the 16S and 23S rRNA regions that are exposed to each reagent [20]. We first used dimethyl A BC Fig. 1. (A) Immunoelectrophoretic analysis of purified EF-P. Purified EF-P protein was visualized with Coomasie Brilliant Blue and detected on immunoblots of the electropho- retic fractions using a specific mouse mAb. The antibody was prepared as described in Experimental procedures. Purified EF-P pro- tein was subjected to isoelectric focusing run in one dimension followed by SDS elec- trophoresis in the second dimension using conditions described previously [23]. (B) Im- munoblots of polyribosomes treated with anti-(EF-P) mAb. The optical density profiles of fractions collected after sucrose-density gradient centrifugation of polyribosomes are shown. The position of the 70S,50S and 30S particles is indicated by arrows. An immunoblot of EF-P, associated with differ- ent fractions of the ribosome, is shown below the ribosome profile. (C) Binding of EF-P to different polyribosome fractions. The density of the immunoblots was deter- mined, as was the area of each peak. The ratio of the density, which is related to the amount of EF-P bound, to the area of the peaks, which is related to the total amount of the ribosome population in that region of the polyribosome profile, is plotted as a function of the percentage of EF-P protein bound to each fraction. Table 1. EF-P modification. MS analysis of tryptic fragments of EF-P protein was carried out as described in Experimental procedures. Table 1 provides information regarding the quality of the search match, including the acquired MS ⁄ MS scan numbers, final candidate search cross-correlation (X-corr) score, the normalized delta cross-correlation score (indicating the difference between the top-ranked and second best match), the preliminary database (SIMS) search score, the matched protein identity, the matched peptide sequence with putative modi- fication sites indicated with an @ symbol adjacent right hand to the target residue, and the predicted modification mass. The average modifi- cation mass, and SD, is also shown. Modification mass, 143.77 ± 0.15. Scan X-corr score DCn SIMS score Protein Sequence (@=modification) Modification mass EFP_115min.2009.2009.2.out 3.3168 0.4493 9 EC4040 Y.AVEASEFVK.P EFP_115min.2147.2147.2.out 2.9467 0.3855 9 EC4040 Y.AVEASEFVK.P EFP_115min.2172.2172.1.out 2.9151 0.3799 108 EC4040 Y.AVEASEFVK.P EFP_115min.2109.2109.2.out 2.905 0.4137 9 EC4040 Y.AVEASEFVK.P EFP_115min.2302.2302.2.out 2.8987 0.3769 9 EC4040 Y.AVEASEFVK.P EFP_115min.2185.2185.2.out 2.7924 0.3979 9 EC4040 Y.AVEASEFVK.P EFP_115min.2048.2048.2.out 2.703 0.4267 9 EC4040 Y.AVEASEFVK.P EFP_115min.2224.2224.2.out 2.7001 0.4626 9 EC4040 Y.AVEASEFVK.P EFP_115min.2523.2523.2.out 3.0763 0.2885 236 EC4040 Y.AVEASEFVKP@GK.G 143.9 EFP_115min.2870.2870.2.out 2.9992 0.2478 214 EC4040 Y.AVEASEFVKPG@KGQAFAR.V 143.6 EFP_115min.2483.2483.2.out 3.6326 0.2885 435 EC4040 Y.AVEASEFVKPGK@.G 143.8 H. Aoki et al. Ribosome interactions with elongation factor EF-P FEBS Journal 275 (2008) 671–681 ª 2008 The Authors Journal compilation ª 2008 FEBS 673 sulfate (DMS), in the presence of borohydride and aniline, to score for A, C and G modifications in the presence or absence of EF-P, and probed the 16S and 23S rRNA. These results guided our further chemi- cal probing with kethoxal (Ke), 1-cyclohexyl-3-(2-mor- pholinoethyl)carbodi-imidemetho-p-toluenesulfonate (CMCT) and diethyl pyrocarbonate (DEP). Recogni- tion sites were identified by primer extension analysis with reverse transcriptase and several synthetic deoxy- oligonucleotide primers. The position of the modified bases was detected by stops or pauses in the progress of reverse transcription of the modified rRNA tem- plate [20,21,24]. Artifact bands, presumably arising from nicks in the template rRNA or from strong sec- ondary structure features, were distinguished from sites of chemical attack by their occurrence in tran- scripts using unmodified control rRNA, which had otherwise been subjected to identical treatment (data not shown). Footprinting experiments were performed with the intact 70S ribosomes in the presence or absence of EF-P protein using probes complementary to 16S rRNA. As shown in Fig. 2, EF-P markedly pro- tects G527, U534 and G537 against CMCT treatment of 70S ribosomes. Also, the reactivity of U531 is enhanced by this treatment. CMCT reacts slowly with G residues [21]; however, DMS protection of this region confirmed these results (data not shown). This region occurs near the G526 streptomycin binding site adjacent to the A-site of the 30S subunit, close to the S12 and mRNA binding sites [25]. Footprinting experiments were also performed on the 23S rRNA modified in the presence or absence of the ribosome-bound EF-P protein. As shown in Fig. 3, EF-P protects A2564, G2524, C2507, G2505, G2502 Table 2. Stoichiometric binding of EF-P to 70S ribosomes and to 30S and 50S subunits. EF-P protein was labeled as described in Experimental procedures. Approximately 240 pmol labeled EF-P were added to 140 pmol 70S ribosomes and incubated for 10 min at 37 °C prior to loading the proteinÆ ribosome complex on the gradi- ents. The samples were centrifuged in a 0–40% sucrose gradient in 10 m M MgCl 2 ,10mM Tris, HCl, pH 7.4 and 30 mM NH 4 Cl for 21 h at 4 °C. Samples were collected and the radioactivity and A 260 values were determined. Particle 70S 50S 30S Particle pmol 18.9 22 17 [ 14 C] EF-P bound pmol 19.7 20.0 14.7 Ratio a 0.959 0.909 0.864 a [ 14 C] EF-P bound ⁄ 70S,50S or 30S. Fig. 2. Primer extension analysis of CMCT-modified 16S rRNA in the presence (+) or absence (-) of EF-P protein was conducted as described in Experimental procedures. The positions of chemical attack were determined using reverse transcriptase and a 22-nucle- otide cDNA 5¢-AGATGCAGTTCCCAGGTTG-3¢ primer complemen- tary to bases flanking the G526 streptomycin binding site. A, T, G, C are dideoxy sequencing lanes. Experiments were performed in triplicate. Ke and DMS treatment of the ribosomes verified the location of the protected bases (data not shown). The data indicate that EF-P protects the 16S rRNA near the A-site (see text). Ribosome interactions with elongation factor EF-P H. Aoki et al. 674 FEBS Journal 275 (2008) 671–681 ª 2008 The Authors Journal compilation ª 2008 FEBS and G2494 in the 23S rRNA against DMS treatment of the ribosome. Significantly, the reactivity of A2572, G2570, G2550 and C2498 is enhanced by the interac- tion of EF-P with 70S ribosomes. It is clear from the data that EF-P markedly protects domain V of the 50S subunit, which is associated with the peptidyl- transferase functions of the ribosome. Most important to the function of the EF-P protein is the fact that most of the molecule’s interactions occur at the aminoacyl-tRNAs (A-site). Indeed, one set of bases protected by interactions of EF-P with the 70S ribosome occurs near the A-loop of the peptidyl- transferase. In addition, the protected bases, A2564 and G2524, map adjacent to the A-site on the 50S sub- unit near the ribosomal 530 loop that is involved in decoding by EF-TuÆGTPÆaminoacyl-tRNA complex, the T-site [26,27]. One of the weakly protected bases, G537, also maps to the T-site of the 30S subunit. It is possible that the T-site represents an alternate weak binding site for EF-P (Fig. 5). To further verify whether EF-P affects the ribosomal A-site, the fMet–tRNA was bound under conditions known to bind aminoacyl-tRNAs to either the ribo- somal A-site or the P-site. The reaction was conducted at 30 °C in the presence of GTP under conditions that foster spontaneous translocation [28]. As shown in Fig. 4A,C,D, GTP markedly stimulates the EF-P- dependent synthesis of fMet–puromycin when the fMet–tRNA is bound to the A-site. This effect is greatly diminished when fMet–tRNA is bound under conditions that bind it to the presumed P-site (Fig. 4B). The oxazolidinone III antibiotic, which impedes binding of fMet–tRNA to the P-site and inhibits translocation [29], interferes with the action of EF-P by preventing the fMet–tRNA substrate from re- binding to the P-site [29]. Oxazolidinone III has little effect when fMet–tRNA is pre-bound to the P-site (Fig. 4B), probably because fMet–tRNA out-competes oxazolidinone III on the P-site. To learn whether fMet–tRNA binding is affected by the action of GTP, fMet–tRNA was bound in the presence of GTP and EF-P for 20 min at 30 °C followed by incubation with puromycin. As shown in Fig. 4C, the reaction proceeds quantitatively. The controls show that the reaction decreases by  70% when fMet–tRNA is bound to the ribosome in the absence of GTP and EF-P, and these reagents are added during the second reaction with puromycin (Fig. 4D). Thus, EF-P may interact with the A-site of the ribosome and may be required to accommodate fMet–tRNA in the P-site of the 70S ribosome. A computer-simulated structure of the 70S ribosome of E. coli was used to fit the protected bases of the EF-P protein relative to the A-, P-, and E-sites occu- pied by their respective tRNAs as well as the mRNA- binding sites (Fig. 5). As shown in Fig. 5, there appear to be two possible binding sites for the protein. One of these occupies the 50S and 30S A-site, whereas the second, weaker binding site, appears to occur on the adjacent T-site of the 70S ribosome. Discussion Biochemical studies have not revealed any effect of EF-P on the initiation reaction. Thus, EF-P is not Fig. 3. Primer extension analysis of DMS-modified 23S rRNA in the presence (+) or absence (–) of EF-P protein. The positions of chemical attack were determined using reverse transcriptase and a 22-nucleotide cDNA primer 5¢-TCTCCAGCGCCACGGCAGATAGG GACC-3¢, complementary to domain V of the 23S rRNA as described in Experimental procedures [20,21,24]. A, T, G, C are di- deoxy sequencing lanes. Experiments were performed in triplicate. The data indicate that EF-P protects domain V on the 23S rRNA, which is close to the A-site of the peptidyltransferase center (see text). H. Aoki et al. Ribosome interactions with elongation factor EF-P FEBS Journal 275 (2008) 671–681 ª 2008 The Authors Journal compilation ª 2008 FEBS 675 required for the binding of fMet–tRNA in the presence or absence of the initiation factors, IF-1, IF-2 or IF-3 [1,7,12]. However, EF-P stimulates the initial rate of translation programmed by poly(rU) when synthesis is initiated by N -acetyl-Phe-tRNA [12] in the absence of initiation factors. Under these conditions, EF-P does not stimulate the synthesis of poly(Phe) from Phe- tRNA dependent on EF-Tu and EF-G [12]. Thus, the EF-P protein may not be required for initiation on 30S subunits or for elongation of protein chains. EF-P is required for synthesis of several fMet-inititated di- peptides [8,12]. Therefore, EF-P may function in the formation of the initial peptides in the protein sequence. Consistent with this idea is the fact that EF-P is found bound to native polyribosomes as well as to 70S,50S and 30S particles (Fig. 1B,C). The EF-P protein occurs in 0.1 copies per ribosome and may dissociate from ribosomes, perhaps after synthesis of the first peptide bond [30]. Here, we present the results of chemical protection footprinting analysis that provide clues concerning the nature of the in vitro associations between rRNA molecules and the EF-P protein. The entire length of the exposed 16S and 23S rRNA molecules on the 70S ribosome was probed with DMS, Ke, DEP and CMCT in the presence or absence of EF-P. A selected number of bases is protected from chemical probes by the complex. In several regions of the rRNA mole- cules, the complex enhanced the reactivity of specific bases, which is interpreted as being due to protein- dependent conformational changes in the rRNA [20]. The evidence presented here indicates that the elon- gation factor, EF-P, binds predominantly to the A-site of 70S ribosomes. A possible second weak binding site Fig. 4. Effect of binding fMet–tRNA to the A- or to the P-site on the activity of EF-P. f[ 35 S]Met–tRNA (36 pmol) was incubated with 70S ribosomes (20 pmol) for 20 min at 30 °C as described previously [22,28] using 4.8 m M (A) or 7.2 mM Mg(Ac 2 ) (B) for bind- ing, presumably to either the A- or the P-site of the ribosome, respectively. EF-P (20 pmol) and puromycin (1 l M) were added and the reaction was continued at 30 °C for 5 min in the presence or absence of 0.1 m M GTP and, where indicated, 50 lM oxazolidi- none III (oxo). (C) EF-P and GTP were added during formation of the fMet–tRNAÆribo- some complex (first reaction) and the reac- tion was carried out for 20 min at 30 °C, prior to the addition of puromycin (second reaction). (D) The initiation complex was formed without GTP or EF-P for 20 min at 30 °C, and GTP and EF-P were added with puromycin and were incubated for 5 min at 30 °C. Ribosome interactions with elongation factor EF-P H. Aoki et al. 676 FEBS Journal 275 (2008) 671–681 ª 2008 The Authors Journal compilation ª 2008 FEBS for the EF-P protein occurs proximal to the A-site on the ribosomal T-site that is the recognition center for the EF-TuÆGTPÆaminoacyl-tRNA on the 30S and 50S subunit. The footprints on the 16S rRNA reside close to the mRNA binding site on the neck of the 30S subunit, adjacent to the G526 streptomycin and the ribosomal protein S12 binding sites (Figs 2 and 5). Interestingly, the structural paralog of EF-P, eIF5-A, which shares strong sequence homology with domains I and II of EF-P, binds to certain mRNAs in a hypusine-dependent manner [31]. Our MS analysis of EF-P indicates that Lys34 of the protein is modified by spermidine, which is a precursor of hypusine in the eIF-5A protein [32]. The model in Fig. 5, in which the orientation of the spermidine residue of EF-P is facing the mRNA-binding pocket of the 30S subunit is con- sistent with this result. The EF-P footprints extend into the 50S subunit and reside close to the L7 ⁄ L12 stalk. The 50 S T-site harbors the ribosomal L12 protein (GAR), which occurs in an open state when the A-site is empty or filled with tRNA [33], but closes upon contact with the EF-Tu ternary complex [33]. It is possible that EF-P binds to the T-site in order to maintain a closed A-site. This may insure that the fMet–tRNA is accommo- dated on the P-site of the 70S ribosome prior to the proof-reading that occurs with the EF-Tu ternary com- plex and the ribosome. The EF-P protein binds stoichiometrically to the ribosome and stimulates accommodation of fMet– tRNA, presumably to the 70S P-site. Thus, it is likely that the protein binds to more than one site on the ribosome depending on the course of synthesis. Most bases protected upon binding of EF-P to the 70S ribo- some appear to reside on the A-site of the 30S and 50S subunits (Figs 3 and 5). It is noteworthy that EF-P protects bases that occur at the A-site of the 50S subunit on 70S ribo- somes (Figs 3 and 5). This is consistent with the EF- P protein being a competitive inhibitor of puromycin [34], an antibiotic known to act at the A-site of the ribosome [35]. Reconstitution experiments, with core particles of the 50S subunit lacking specific 50S pro- teins, revealed that L16 is essential for the action of the EF-P protein [36]. The crystal structure of the ribosome, with bound aminoacyl-tRNA, indicates that L16 occurs at the A-site of the 50S subunit [37]. Furthermore, the L16 protein binds aminoacyl-tRNA [37] (and unpublished observations). The tRNA-like shape of the EF-P protein is in keeping with its interactions near the A-site of the 70S ribosome and its subunits. The binding of EF-P at the A-site may have important consequences for proper decoding of the mRNA transcript. For example, by binding to the A-site, EF-P may prevent the incorrect position- ing of the fMet-RNA into the A-site of the 50S sub- unit. The binding site of EF-P to domain V is of special importance because EF-P is predicted to enhance elon- gation by influencing in some manner the peptidyl- transferase center [12]. Several bases indicated in the protection analysis (Fig. 3) demonstrate that EF-P interacts close to the A-site of domain V of the 23S rRNA. The X-ray diffraction patterns of the 70S ribosomes of Thermus thermophilus, at 5.5 A ˚ reso- lution, reveal that the binding site of the three tRNAs is adjacent to the binding site of the elongation factors [37]. Thus, the incoming aminoacyl-tRNAÆGTPÆEF-Tu complex must be adjusted to the A-site in a position where a peptide bond can be formed. By being at the A-site, EF-P may prevent the spurious entrance of aminoacyl-tRNAs, or of deacyl-tRNAs, which do not occur in the ternary complex with EF-Tu, from prema- turely entering the A-site prior to the proofreading steps which precede accommodation of the incoming aminoacyl-tRNA into the peptidyltransferase active site. Fig. 5. Computer-simulated 3D structure of the 70S ribosome of E. coli showing the approximate location of the EF-P-binding site on the 70S ribosome. Bases protected against chemical modification by interactions with EF-P are shown in red; bases enhanced by the interactions with EF-P are shown in yellow. The positions of the exit (E) and peptidyltRNA (P) site on the 70S ribosome are shown. EF-P (light green) clearly binds near the A-site of the 30S and 50S subunits, as well as to the T-site of the ribosome. The probable spermidine-binding site (on Lys34 of EF-P protein) is shown in orange and points towards the 30S subunit (see text). The coordi- nates for the E. coli ribosome were from Schuwirth et al. [43]. The protections observed were also found to neighbor the A-site of the 70S ribosomes of T. thermophilus determined by X-ray diffraction at the 5.5 A ˚ resolution [37]. H. Aoki et al. Ribosome interactions with elongation factor EF-P FEBS Journal 275 (2008) 671–681 ª 2008 The Authors Journal compilation ª 2008 FEBS 677 We suggest that EF-P interacts with the A-site of the peptide-bond-forming center of the large ribosomal subunit and with the decoding center of the small sub- unit. Thus, the binding site may span both subunits. The oligonucleotide domain of the EF-P protein, which harbors spermidine, may bind near the mRNA- binding site (Fig. 5), while the hydrophobic residues around the central loop of the molecule might be adja- cent to the peptidyltransferase center. Most important to the function of the EF-P protein is the fact that the interactions of the molecule are in close proximity to the peptidyltransferase center of domain V near the positions of the A-site 3¢-CCA-termini of the bound tRNAs (Figs 3 and 5). The footprints of the EF-P protein on the 16S rRNA are adjacent to the G526 streptomycin-binding site [22,35] and the ribosomal protein S12 (Figs 2 and 5). Streptomycin is a potent inhibitor of EF-P-medi- ated synthesis of peptide bonds [12]. Streptomycin inhibits translation by increasing the error rate of syn- thesis and interfering with the proofreading mecha- nisms of the ribosome [25,35]. When a cognate tRNA binds to the A-site, the 30S subunit undergoes a conformational change from an open to a closed form [25]. Some mutations that affect accuracy either prevent or induce this conforma- tional transition [25]. Streptomycin, for example, stabi- lizes the closed form and induces errors in translation. By contrast, certain mutations in S12 to streptomycin resistance or dependence destabilize the closed form [25]. Thus, by binding near the A-site, EF-P may prevent the aminoacyl-tRNA in the EF-TuÆGTPÆaminoacyl- tRNA complex from prematurely entering the A-site prior to the proofreading functions carried out by the ribosome [25–27]. This interaction would also help poise the fMet–tRNA on the P-site of the 50S subunit and may also result in increased accuracy of amino- acyl-tRNA selection. Hydrolysis of GTP results in ejection of EF-TuÆGDP from the ribosome and accom- modation of the aminoacyl-tRNA into the A-site [26,27]. A-site-bound aminoacyl-tRNA is predicted to displace the EF-P protein from the ribosome. Thus, one EF-P molecule would be used for each successfully initiated round of translation. The interactions of EF-P with the ribosome are likely to result in rearrangements of the ribosomal subunits that are essential for the transition between the initiation and elongation stages of protein synthe- sis. The conservation of this protein throughout spe- cies and its obligatory requirement during synthesis befit the essential nature of these functions in transla- tion. Experimental procedures Materials The materials used were as described in Xu et al. [5] and [29]. RNA modification and primer extension analysis The 70S ribosomes (8 pmol) were modified using DMS, DEP, Ke or CMCT in the presence or absence of 20 pmol EF-P protein. Adenines and cytosines were determined as described previously [20,21]. The N-7 position of guanine was detected with DMS using sodium borohydride and ani- line to induce strand scission [21]. The DNA sequence was performed by a modification of the double-strand dideoxy chain termination method of Sanger et al. with modified T7 DNA polymerase and [ 35 S]dATP[aS] [20,21,24]. Effect of EF-P binding on the chemical protection footprints of 70S ribosomes To locate the bases in 16S and 23S RNA on 70S ribosomes that are in contact with EF-P, EF-P was first bound stoi- chiometrically to the ribosome. Each of the synthetic oligo- nucleotide primers was annealed to the rRNA in the complex and was extended with reverse transcriptase in the presence of [ 35 S]dATP[aS] and the other three (unlabeled) deoxynucleotide triphosphates. The labeled DNA tran- scripts were resolved on a DNA sequencing gel along with four dideoxy sequencing lanes on the same gel to help iden- tify the modified bases. The entire length of the 16S or 23S rRNA on the 70S ribosomes was probed with DMS in the presence or in the absence of the EF-P protein. Primers complementary to sequences at  200-bp intervals were then annealed to the ribosome [20]. Based on the results with DMS, several different primers were utilized to further study the specificity of the regions protected against chemi- cal attack by Ke, CMCT and DEP. Primers to the 30S sub- unit spanned the streptomycin binding sites at G526 and at A915 [25]. Two additional primers were used to probe the decoding region of the 16S rRNA. Two primers were used to probe domain II of 23S rRNA; three primers were used to probe domains IV and V and one primer was used to probe domain VI of the 23S rRNA molecule. Preparation of 70S ribosomes, 30S and 50S subunits Ribosomes and subunits were isolated from E. coli MRE- 600 mid-log cells at 4 °C as described previously [22]. Briefly, cells (100 g) were broken by grinding with 100 g alumina (Alcoa Inc., Pittsburgh, PA, USA) and suspended in buffer A (10 mm Tris ⁄ HCl, pH 7.4, 30 mm NH 4 Cl, 1mm dithiothreitol, 6 mm Mg(Ac) 2 ), DNase (RNase-free; Ribosome interactions with elongation factor EF-P H. Aoki et al. 678 FEBS Journal 275 (2008) 671–681 ª 2008 The Authors Journal compilation ª 2008 FEBS 1.0 lgÆmL )1 ) was added and the mixture incubated for 5 min at 4 °C. Unbroken cells and debris were removed by two successive centrifugations for 20 min at 30 000 g. The resulting supernatant was centrifuged at 78 000 g for 18 h and the ribosomal pellets were suspended in buffer A con- taining 0.5 m NH 4 Cl and the crude ribosomal suspension was gently stirred for 2 h. The crude ribosomal suspension was layered on sucrose density gradients (0–40% sucrose in buffer A) and centrifuged at 78 000 g for 18 h. Fractions containing the 70S ribosomes were combined and were cen- trifuged for 24 h at 60 000 g. The ribosomal pellets were suspended in 2–3 mL buffer A containing 0.5 m NH 4 Cl, the suspension was stirred for 2 h at 4 °C and was subjected to a second 0–40% sucrose density gradient centrifugation step as above at 64 000 g. Absorbance at 260 nm was used to identify the ribosomal fractions and the 70S peak was pooled and centrifuged at 37 000 g for 18 h. Ribosomes were collected and suspended in buffer A at a concentration of 20 mgÆmL )1 and stored at )80 °C. Isolation of EF-P The EF-P protein was assayed based on its ability to stimu- late ribosomes to synthesize fMet–puromycin in the pres- ence of 1.0 lm puromycin and the absence of organic solvents [1] using f[ 35 S]Met–tRNA bound to ribosomes as described previously [22,38]. The EF-P protein was purified as described previously [12]. Briefly, ribosomes were isolated as described above and washed once with 0.5 m NH 4 Cl in buffer A containing 6mm Mg(Ac) 2 . The ribosomal wash, containing the EF-P protein, was sequentially purified first through a column of QAE–Sepharose, followed by hydroxylapatite, Sephacryl S-300 and Mono-Q columns using FLPC. Fractions were assayed after each step to locate the protein. The purified protein was concentrated and was stored at )80 °C. Protein concentration was determined using the method described by Bradford [39]. Preparation of mouse mAbs to EF-P mAbs to the purified EF-P protein were raised using nude mice. This study received ethical approval by the University of Toronto Animal Care Committee, which is in full com- pliance with the Guidelines of the Canadian Council on Animal Care and the Regulations of the Animals for Research Act. Aliquots of the EF-P (1 mgÆmL )1 ) in buf- fer A were injected into the mice intravenously. The immu- nization schedule was 5 lg of the protein on day 1, 10 lg on day 8, 20 lg on day 12 and 25 lg on day 16. After a two-week rest, mice received a final 25 lg injection of the EF-P protein. Three days after the final injection, blood was obtained through the orbital vein. Three days after this, the cells were harvested and fused with HAT-sensitive mouse myeloma cells. The samples were tested first for anti- body production using dot blots. Positive clones were ana- lyzed on western blots. Radioactive chemical labeling of the EF-P protein The EF-P protein (700 pmol) was dissolved at 1 °Cin 0.1 m Na borate buffer (pH 9.0), 300 mm KC1 and 5 mm b-mercaptoethanol. The [ 14 C]formaldehyde (50 mCiÆmm )1 ) was added in the above buffer to the EF-P protein, mixed for 2 min. prior to the addition of 8 lg Na borohydride to stop the reaction. The labeled EF-P was dialyzed against 10 mm HEPES buffer, pH 7.4, 10 mm b-mercaptoethanol to eliminate the unreacted [ 14 C] formaldehyde. Tandem MS Proteomic analysis of the samples was performed by microcapillary electrospray LC-MS ⁄ MS. Briefly, an aliquot of the EF-P protein was suspended in 100 mm NH 4 HCO 3 ⁄ 1mm CaCl 2 buffer, pH 8.5 and digested by trypsin overnight at 37 °C using immobilized trypsin Porous beads (PerSeptive Biosystems, Framingham, MA, USA). The digested peptides were then fractionated on a 7.5 cm (100 lm ID) reverse-phase C 18 capillary column attached inline to a Thermo Finnigan LCQ-Deca quadrupole ion trap mass spectrometer. The entire digested sample was loaded as described previously [40] and the peptides eluted by ramping a linear gradient from 2 to 60% solvent B over 90 min. Solvent A consisted of 5% acetonitrile, 0.5% acetic acid and 0.02% heptofluorbutyric acid and solvent B con- sisted of 80 : 20 acetonitrile ⁄ water (v ⁄ v) containing 0.5% acetic acid and 0.02% heptofluorbutyric acid. The flow rate at the tip of the needle was set to 300 nLÆmin )1 by program- ming the HPLC pump and use of a split line. The mass spectrometer cycled through four scans as the gradient pro- gressed. The first event was a full mass scan followed by three tandem mass scans of the successive three most intense precursor ions. Precursor ions (400–2000 m ⁄ z) were sub- jected to data-dependent, collision-induced dissociation with dynamic exclusion enabled. The spectra were searched against UniProt protein sequences downloaded from the European Bioinformatics Institute using the sequest com- puter algorithm [41]. Precursor mass tolerance was set to 3 Da (with daughter mass ion tolerance set to the default of 0), enabling fully tryptic enzyme status, with single site missed cleavages tolerated. The statistical probability of each primary match was assessed using the statquest algo- rithm [42], with candidate peptides filtered using a strict 95% confidence cut-off to minimize false positives. Acknowledgments We thank NSERC of Canada for financial support. We thank Jennifer Yang and Ivona Kozieradsky for H. Aoki et al. 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