The intergenic IRES of Cricket Paralysis Virus (CrPV-IRES) forms a tight complex with 80S ribosomes capable of initiating the cell-free synthesis of complete proteins in the absence of initiation factors. Such synthesis raises the question of what effect the necessary IRES dissociation from the tRNA binding sites, and ultimately from all of the ribosome, has on the rates of initial peptide elongation steps as nascent peptide is formed. Here we report the first results measuring rates of reaction for the initial cycles of IRES-dependent elongation. Our results demonstrate that 1) the first two cycles of elongation proceed much more slowly than subsequent cycles, 2) these reduced rates arise from slow pseudo-translocation and translocation steps, and 3) the retarding effect of ribosome-bound IRES on protein synthesis is largely overcome following translocation of tripeptidyl-tRNA. Our results also provide a straightforward approach to detailed mechanistic characterization of many aspects of eukaryotic polypeptide elongation.DOI:http://dx.doi.org/10.7554/eLife.13429.001 Inside cells, machines called ribosomes make proteins using instructions carried by molecules of messenger RNA (or mRNA). The ribosomes bind to the mRNA and then move along it to assemble the proteins in a process called translation. The first step of translation – when the ribosome binds to the mRNA – is known as initiation. In human and other eukaryotic cells, initiation mainly occurs through a mechanism that requires many proteins called initiation factors to recruit the ribosome to a cap structure formed at one end of the mRNA.When viruses infect cells, they hijack the ribosomes of the host cell to produce large quantities of viral proteins. However, unlike their host cells, many viruses use a different pathway to initiate translation of their mRNAs. The mRNAs of these viruses have regions known an internal ribosome entry sites (IRESs) that host cell ribosomes can bind to instead.After initiation, the ribosome progressively assembles the building blocks of proteins (amino acids) into a chain until the new protein is complete. Molecules called transfer RNAs bind to individual amino acids and bring them to the ribosome. Previous research has shown that, prior to initiation, IRESs on Cricket Paralysis Virus mRNAs bind to the ribosome and occupy sites where transfer RNAs would normally bind. However, it was not clear how this affects the elongation process. Zhang et al. now address this question using a cell-free system that allowed them to recreate and observe translation outside of the normal cell environment.Zhang et al. found that the binding of an IRES to a ribosome slows down the early steps of elongation. A likely explanation for this is that the IRES elements have to be displaced from the ribosome before the incoming transfer RNAs can occupy the three tRNA sites. However, as elongation progresses, the effects of the IRES elements are overcome and the pace of elongation increases significantly. Zhang et al.’s findings provide a convenient approach that could be used for future studies of elongation. This approach could also help researchers find out how abnormalities in translation contribute to human diseases, including muscle-wasting disorders.DOI:http://dx.doi.org/10.7554/eLife.13429.002 Initiation of protein synthesis in eukaryotic cells proceeds via two well-established pathways. The cap-dependent pathway involves recognition of 7-methyl-guanosine at the 5’-terminus of mRNA by a preinitiation complex of 40S ribosomal subunit and a host of initiation factors prior to a scanning step that results in initiator aminoacyl-tRNA(aa-tRNA) pairing with a cognate start codon, followed by 60S binding to form the 80S initiator complex . The secd ~ 10 nM) to the 80S ribosome (7) requires removal of the IRES from the last of the three tRNA binding sites. In the work reported below, we demonstrate first, that the initial elongation steps are indeed quite slow and are limited by the translocation step of the elongation cycle, and second, that the rate of elongation accelerates following translocation of tripeptidyl-tRNA to the P-site.CrPV-IRES binds with high affinity  to the 80S·CrPV-IRES (80S·IRES) complex . These results indicate that, consistent with recent results of 1 and 2 is shifted in the presence of eEF2·GTP, such that approximately half of 80S·IRES is present as 2.Phe-TC binding to 2, resulting in the formation of Structure 3, accounts for the rapid phase in the red and black traces. Further formation of 3 is limited by the slower rate of 1 to 2 conversion. Although added eEF2·GTP decreases all three apparent rate constants, the effect is much greater on k-1 (~50-fold reduction) than on either k1 (~twofold reduction) or k2 (~fourfold reduction). The near identity of the red and black traces, performed at different eEF2·GTP concentrations, suggests that this factor interacts with both 1 and 2, with a dissociation constant significantly less than 1 µM. The large inhibitory effect of eEF2·GTP on k-1 is consistent with its role as a translocase, and with recent results demonstrating that a principal role of EF-G, the prokaryotic equivalent of eEF2, is to inhibit back-translocation  on the rate of formation of 4 from 1   proceed in remarkably similar fashion, each showing biphasic behavior with a rapid phase accounting for 65 ± 10% of reaction proceeding with a t1/2 of ~6–9 s and a slower, minor phase proceeding much more slowly (t1/2 ~220–240 s), possibly corresponding to defective ribosomes. Reactions involving formation of two peptide bonds, as in the conversion of 4 to 9 or 7 to 12 are well approximated as single phase reactions with t1/2 values of 90–110 s. Conversion of 4 to 9 proceeds via Steps 4 – 8, allowing the t1/2 value for the translocation Step 6 to be estimated as 84 s, from the difference between the t1/2 value for the 4 to 9 reaction and the sum of the t1/2 values for the 4 to 6 and 7 to 9 reactions (major phases). Similarly, the t1/2 value for the translocation Step 9 can be estimated as 110 s from the difference between the t1/2 value for the 7 to 12 reaction and the sum of the t1/2 values for the 7 to 9 and 10 to 12 reactions. Since the di-, tri- and tetrapeptides synthesized in the results reported in 1/2 values depend on the not unreasonable assumption that the identities of the tRNAs undergoing translocation do not have a major influence on the translocation rate. With this caveat, the results presented in 4 to 10.Using ribosomes programmed with the appropriate coding sequence mutants  and [35Sis (TLE) . For Pheis (TLE)  we prefoucture 7  and PheVcture 10 . In all 35S]-Met-tRNAMet is able to cosediment with the ribosome following mixing of [35S]-Met-TC with structures 4, 7, or 10. This strategy was successful for [35S]-Met–TC reaction with structure 7  and peptide bond formation . They also provide a clear indication that, within Structures 8, 9, 11 and 12, Met-tRNAMet, PheLysMet-tRNAMet, and PheValLysMet- tRNAMet, whenbound to the A-site, efficiently cosediment with ribosomes, which is typical for A-site bound tRNAs in conventional (non-IRES) elongation complexes  from the peptide formation step  we also employed a rapid mixing and quench assay to determine the rates with which [omplexes .35S]-Met–TC reaction with structure 4 (containing P-site bound Phe-tRNAPhe), the [35S]-Met-TC cosedimentation rate is much slower than the dipeptide formation rate  is fully recovered by cosedimentation. As a result, the cosedimentation assay does not permit estimation of the t1/2 values for Steps 4 and 5. It is possible that the lability of the A-site tRNAs in structures 5 and 6 is due to IRES binding to the E-site, which is absent in structures 8, 9 and 11, 12, and may reflect an allosteric A-site: E-site interaction. Evidence for allosteric A-site/E-site interactions has been presented for both bacterial and eukaryotic ribosomes  than it reacts with P-site bound PheValLys-tRNALys(t1/2 76 ± 16 s). The corresponding t1/2 value for puromycin reaction with PheValLys-tRNALys undergoing translocation from the A- to P-site is 170 ± 30 s. This increase of approximately 100 s for translocating PheValLys-tRNALysvs. translocated PheValLys-tRNALys closely matches the t1/2 value of 110 ± 30 s estimated above for the translocation of tripeptidyl-tRNA (Met (Structure 13)are indistinguishable from one another  and PheValLysMet-tRNAMet . These rates, while consistent with those reported by others for puromycin reaction with eukaryotic P-site bound Met-tRNAMet  vs. tetrapeptidyl-tRNA (Step 12), leads to the prediction that synthesis of a longer peptide that required the tripeptidyl-tRNA translocation step (Step 9) would proceed significantly more slowly than synthesis not requiring this step.Above we have demonstrated that, under our conditions, aa-tRNA binding and peptide bond formation proceed with an overall tLys (Structure 7) vs. P-site bound PheLysVal-tRNAVal (Structure 10)  . Indeed,The results presented in this paper constitute the first time that rates of reaction have been determined for the initial cycles of IRES-dependent elongation. They demonstrate quite clearly that the first two cycles of elongation proceed much more slowly than subsequent steps, and that these reduced rates arise from slow, rate-determining, pseudo-translocation and translocation steps. Translocation during the first elongation cycle (Step 6) clearly requires displacement of the IRES from the E-site, so it is not unexpected that it would be slow. Less predictable is the slow translocation in the second elongation cycle, (Step 9) after the IRES structure has, presumably, already left the E-site . The slo4 in which aa-tRNA binds to the P-site. However, it is clear from the anisotropy experiment conducted in the absence of added eEF2·GTP (3)does not require eEF2·GTP. This is easily understood as an example of Le Chaltelier’s principle, in which the equilibrium between Structure 1 (closed A- site)and Structure 2 (open A- site), which strongly favors Structure 1, is pulled to the right by aa-tRNA binding. Preincubation with eEF2 also shifts the equilibrium to the right, leading to an initial rapid phase of reaction with Phe-TC . Although this rate is essentially identical to that reported for tripeptide synthesis in a cap-dependent yeast-based in vitro translation system which requires five initiation factors and eEF3 in addition to eEF1A and eEF2 (-1)  . There iEF2 (-1) . It has EF2 (-1) . The sloEF2 (-1) .Detailed mechanistic characterization of many aspects of eukaryotic polypeptide elongation has been held back by the lack of a convenient system for its study. The very simple in vitro IRES-dependent elongation system described here should be useful in overcoming this limitation. As one example, it is generally assumed, based on extensive structural similarities , that eEPhe was available to us and the appropriate tRNAAla acceptor was not. The vectors encoding the PheMet, PheValMet, PheValLysMet, and PheLysValArgGlnTrpLeuMet were generated by PCR insertion of corresponding sequences  according to the manufacturer. For in vitro transcription of short-length mRNAs, the mutated IRES plasmids were linearized with NarI, which cleaves 33 nt downstream of the ATG start codon of the luciferase coding region.For in vitro transcription of full-length mRNA for the Luciferase assay, the WT and mutated Phe-IRES plasmids were linearized with In vitro translation of firefly luciferase with WT and mutated F-IRES mRNA (1 μg in 50 μL of reaction mixture) was performed using the Flexi Rabbit Reticulocyte Lysate System (Promega) according to the manufacturer. IRES mRNA was omitted in the control reaction. Fluc activities  were detA. salina) 80S ribosomes were prepared from dried, frozen cysts as previously described , was prepared as previously described (Phe was purchased from Sigma. Other isoacceptor tRNAs were prepared from bulk tRNA (Roche) from either E. coli  or yeast  by hybridization to immobilized complementary oligoDNAs, as described  at 37°C. For the preparation of ternary complexes  and 80S·IRES complexes containing either Phe-tRNAPhe or peptidyl-tRNA bound in the P-site, buffer 4 was supplemented with 1 mM GTP and 1 mM ATP. All TC complexes were prepared by incubating the relevant charged tRNA  with eEF1A (8 μM) for 5 min. 80S·IRES complexes were formed by incubation of shrimp 40S (0.8 µM) and 60S (1.6 µM) subunits with the appropriate IRES (2.4 µM) for 5 min. 80S·IRES complexes containing Phe-tRNAPhe or peptidyl-tRNA bound in the P-site were formed by mixing 80S·IRES complexes (0.8 µM) with 1 μM eEF2 and the appropriate TCs (1.6 µM for each) for 15–40 min. To determine radioactively labeled aa-tRNA binding stoichiometries, 40 µL samples were subjected to ultracentrifugation at 4°C  for 40 min through a 1.1 M sucrose cushion. Excess bacterial 30S bacterial ribosome subunits (600 pmol/15 ± 5 µL) were added as carrier to enhance pelleting and allow facile calculation of complex recovery. Control experiments carried out in the absence of IRES or of both IRES and 80S ribosomes demonstrated that only negligible amounts of labeled peptidyl-tRNA cosedimented due to binding to 30S subunits ·eEF1A·GTP ternary complex was rapidly mixed with 80S·FVKM-IRES complex in the presence or absence of eEF2·GTP using a KinTek stopped-flow spectrofluorometer model SF-300X. Proflavin labeled Phe-tRNAPhe was excited at 462 nm and monitored using a pair of 495 nm long-pass filters. A T-shape configuration was utilized such that instrument-specific polarizers were attached to both the excitation and the two emission light paths. In each independent measurement, 15–20 shots (rapid mixing of samples) were averaged to provide the time course of anisotropy change. The g-factor and anisotropy value were calculated using the instrument software as described (Phe-tRNAescribed . Experim3H]-Phe-TC in the presence of eEF2·GTP in a KinTek Corporation RQF-3 Rapid Quench-Flow Instrument the reaction mixture was quenched at various times with 0.5 M MES buffer (pH 6.0), and the stoichiometry of ribosome-bound [3H]-Phe-TC was determined by ultracentrifugation as described above for complex characterization. Similar procedures were used to determine the kinetics of [35S]-Met-TC binding to 80S·IRES complexes containing Phe-tRNAPhe or peptidyl-tRNA in the P-site (80S·IRES complex (0.8 μM) with no tRNA bound  was rapie P-site .Phe or the appropriate peptidyl-tRNA in the P-site, prepared using the standard procedure (see Complex preparations above), were rapidly mixed with [35S]-Met-TC (1.6 µM) and additional TCs as required  in a KinTek Corporation RQF-3 Rapid Quench-Flow Instrument, and the reaction mixture was quenched at various times with 0.8 M KOH. [35S]-Met-containing peptide was released from tRNAMet by further incubation at 37°C for 3 hr. The pH of the samples were adjusted with acetic acid to pH 2.8, lyophilized, suspended in water, and centrifuged to remove particulates, which contained no 35S. The supernatant was analyzed by thin layer electrophoresis as previously described  and additional TCs as required , for various times followed by quenching with 0.5 M MES (pH 6.0) buffer. PheLysValArgGlnTrpLeuMet octapeptide synthesis was measured by [35S]-Met cosedimentation with 80S·FKVRQWLM-IRES complexes. Over the time scale of these measurements  were mixed with -Lys-TC for one minute. The resulting complex was then reacted with puromycin.80S·FVKM-IRES complex with FVK-tRNA pre-bound at A-site was formed by incubating the 80S·FVKM-IRES complex containing FV-tRNA at the P-site , purified as described above, was incubated with 0.8 μM [3H]-Lys-TC and eEF2.GTP (1.0 µM) for 15 min at 37°C. The resulting complex was then reacted with puromycin. Procedure 2: 80S·FVKM-IRES complex (0.8 µM) was preincubated for 15 min with eEF2·GTP (1 µM) and Phe-TC, Val-TC, and [3H]-Lys-TC . The resulting complex was then reacted with puromycin.Two procedures were employed, which yielded equivalent results. Procedure 1: 80S·FVKM-IRES complex containing FV-tRNA at the P-site . The resulting complex was then reacted with puromycin.80S·FVKM-IRES complex (0.8 µM) was preincubated for 15 min with eEF2·GTP (1 µM) and Phe-TC, Val-TC, Lys-TC and [Val bound in the P-site. This complex was then mixed for 1 min with ([3H]-Lys-TC (1.6 µM) to form PheValLys-tRNALys bound in the A-site, which was then reacted with puromycin in the presence of additional added eEF2·GTP .80S·FVKM-IRES complex (0.8 µM) was preincubated for 15 min with eEF2·GTP (1 µM) and Phe-TC and Val-TC, both present at 1.6 µM, yielding PheVal-tRNALys bound in the P-site. This complex was then mixed for 1 min with ([35S]-Met-TC (1.6 µM) to form PheValLysMet-tRNAMet bound in the A-site, which was then reacted with puromycin in the presence of additional added eEF2·GTP .80S·FVKM-IRES complex (0.8 µM) was preincubated for 15 min with eEF2·GTP (1 µM) and Phe-TC, Val-TC, and Lys-TC , yielding PheValLys-tRNA In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by John Kuriyan (Senior editor) and three reviewers, one of whom, Alan Hinnebusch, is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled "One, two, three, go – initiating polypeptide elongation in an IRES-dependent system" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary of work:Phe binding to either the A- or P-site, and to allow incorporation of labeled Met-tRNAMet to monitor production of FM, FKM, FVKM, and FKVRQWLM synthesis. Sedimentation of complexes through sucrose gradients was used to assay stable binding of Met-tRNA or peptidyl-tRNAs containing labeled Met to the ribosome. The anisotropy results in Phe binding to the A site in a biphasic reaction. The sedimentation assay indicates that the second pseudotranslocation step, in which Phe-tRNA is translocated from the A to the P site is much slower than the combination of the first pseudotranslocation plus Phe-tRNAPhe binding to the A site. They go on to use a rapid mixing and quench assay to measure rates of FM, FKM, and FKVM synthesis, with detection and quantification of product by thin layer electrophoresis (TLE), using the appropriate pre-formed substrates for addition of labeled Met to Phe-tRNAPhe or the relevant peptidyl tRNA. Results in This paper conducts in vitro experiments using a purified system to study the rates of initial oligopeptide synthesis by an 80S·CrPV-IRES complex. The results suggest that the first two elongation cycles involved in producing a di- or tripeptide are slow and are limited by the translocation step, and that the rate of elongation accelerates following translocation of tripeptidyl-tRNA to the P-site (the second true translocation event). The IRES was suitably modified to replace the 1st Ala codon with a Phe codon, allowing fluorescence anisotropy measurements of the rate of proflavin-modified Phe-tRNAEssential revisions:The following issues raised in the referees' comments shown below need to be addressed in the revised manuscript.Ref. #1: All of the requests for clarification of reaction conditions or interpretations of results should be addressed with appropriate revisions of text.Ref.#2: The request for complete details about fitting the kinetic data in comment #1 should be honored.As described in comment #2, the need for modifying the IRES sequence should be more explicitly stated in Materials and methods, as it would have been technically feasible to carry out all experiments with the WT sequence. In addition, it is important to compare the rates of IRES-mediated translation given by the native sequence versus your modified IRES sequence either in a cell-free extract or in your reconstituted system.To respond to points 3. & 4., it would be sufficient to revise the text to remove statements about allostery; for point 5, to explain why you monitored the puromycin reaction as you did in 1/2 of step 1. You must also discuss the discrepancy with the previous results, particularly those of Ruehle et al.Ref. #3: Points 1.-3 concern your conclusion that eEF2 does not stimulate the rate of TC binding in step 2 and rather only shifts the equilibrium in step 1 towards structure 2, which is at odds with other published work. As this is a hypothetical interpretation of the biphasic kinetics in You should clarify the issues raised in points 4-6 with the appropriate revisions of text.Original reviews:Reviewer #1:The results are significant in providing strong evidence that the two pseudotranslocation steps , as well as peptidyl-Met-tRNAMet binding to the P-site (Structures 10 and 13), efficiently…", the clarity could be improved by mentioning the specific peptidyl-tRNAs by their amino acid compositions for each of the structures, as "peptidyl-Met-tRNAMet" is imprecise nomenclature.1) In the second paragraph of the subsection “Rates of oligopeptide formation and Met-tRNAMet cosedimentation”: Also, it's not perfectly clear whether the instability of Met-tRNA binding to the A-site observed for structure 3 and 5, and FM-tRNA to structure 6 using the co-sedimentation assay is unusual; whereas stable binding of both Met-tRNA and peptidyl-tRNA to the A-site in structures 8, 9, 11-12 is typical of A-site-bound ligands in conventional (non-IRES) elongation complexes; and this distinction needs to be carefully spelled out.2) In the subsection “Rates of oligopeptide formation and Met-tRNA3) In the second paragraph of the subsection “Translocation of tetrapeptidyl-tRNA (Step 12) is much more rapid than of tripeptidyl-tRNA (Step 9)”: requires citation of a figure for puromycin synthesis rates.4) In the second paragraph of the subsection “Translocation of tetrapeptidyl-tRNA (Step 12) is much more rapid than of tripeptidyl-tRNA (Step 9)” and 5) In the last paragraph of Results: more detail is required to understand how binding of peptidyl tRNA harboring labeled Met is distinguished from Met-tRNA binding to the A-site in these assays.6) Error estimates have not been defined in any of the figures or tables and this needs to be rectified.Reviewer #2:The paper by Cooperman et al. provides a first kinetic description of the initial steps of translation by eukaryotic ribosomes after the initiation at an IRES. There are very few papers on mechanistic aspects of eukaryotic translation in general and the present work provides very interesting, significant insights. It is a real challenge to obtain clean kinetic data for the eukaryotic system and this paper provides valuable information on the kinetic properties of eukaryotic ribosomes. The technical quality of the data is high  and the conclusions are warranted. My only reservation is that many technical aspects are not clearly described. Given that this paper delineates new kinetic approaches to study eukaryotic translation, it is particularly important to provide a very detailed description of methods. This really has to be carefully revised. I personally do not like the title, because it is not informative at all; a more rigorous title would be better.Detailed comments:1) Fitting of the kinetic data in this paper is mostly non-exponential and would require fitting to a model by numerical integration. This part is completely missing in Materials and methods. This must be described in all details required to understand and reproduce the calculations.35S]Met; in this case one could use the native sequence. The need for modifying the sequence should be more explicitly stated in Materials and methods. Clearly, the whole set of experiments cannot be repeated with Ala as a 1st amino acid; however, it would be highly desirable to compare IRES-mediated translation with a native sequence and a modified sequence in the cell-free translation system .2) Among all potential criticism of the experimental system , I am particularly worried about the altered coding sequence. It is not clear why the native Ala codon was replaced by Phe, as unlabeled amino acids are used anyway and the tRNAs are prepared by a method which should readily yield tRNA(Ala), i.e. one could label tRNA(Ala) with Prf . This means that in principle one could use the native sequence. Furthermore, the measurements and rate determinations would be even more straightforward if [14C] and [3H]-labeled amino acids were used instead of Met (at least to some extent) even in the absence of the mRNA. Are there controls for that?7) In the subsection “Complex preparations. TCs and various 80S·IRES complexes”. 30S subunits were added to the centrifugation assays as a carrier. However, 30S subunits can bind [8) 35S]Met-TC”. My expectation is that in the absence of the preceding Lys-TC Met should not be incorporated at all. This has to be clarified.9) 10) In the subsection “Kinetic measurements”, "averages of 2-4 independent determinations" – do the authors mean technical replicates or independent experiments ?Reviewer #2 :The statistical information is appropriate except for the clarification needed in the subsection “Kinetic measurements” (point 10 in the review).Reviewer #3:Zhang and colleagues have investigated the effect of the CrPV IRES RNA on the first rounds of translation elongation including the initial pseudo-translocation steps. They have performed for the first time experiments to determine the rates of the various main steps of the elongation cycles. They show that the IRES initial significantly slows down the translocation steps are down until the tetrapeptide stage is reached. The CrPV is an important minimal model system for translation initiation in eukaryotes The results are novel and interesting. However, the taking into account the following points is recommended before the paper can be published.1) According to 2) The authors state that k-1 is much smaller than k2 and was assumed to be negligible. How has this been determined? In fact, a toeprint signal indicative of translocation cannot be obtained by incubation of binary 80S-IRES complexes with eEF2 alone, but requires an A-site ligand, too . This suggest that the translocated complex (structure 2) is kinetically labile and contradicts the present statement that the back-translocation is negligible. Again, the authors should do experiments, e.g. toeprinting, to provide additional evidence that pre-incubation with eEF2-GTP shifts the equilibrium between structures 1 and 2 from 95:5 to 50:50 as stated.1/2 of step 1. How this is possible? Do the authors propose that k-1 is becoming smaller, i.e. that eEF2 slows down back-translocation without accelerating forward translocation?3) The explanation about the effect of eEF2 on the equilibrium between structure 1 and 2 (Discussion) is curious. They propose that eEF2 shifts the equilibrium towards structure 2 at the same time do not measure a significant effect on the t4) Can the authors rule out that back-translocation or peptidyl-tRNA drop off influences the following steps towards tetrapeptide synthesis?Additional points:5) The first sentence of the paper "Initiation of protein synthesis in eukaryotic cells proceeds via two well-established pathways" is to some extent misleading. As the authors acknowledge later factor requirement of different IRES RNAs is diverse and the structure and mechanism of different IRES RNAs is also different. So there are several pathways leading to internal initiation.6) The peptide coding sequence is not really part of the IRES. Therefore, it is misleading to talk about mutant IRES, when the changes are in the ORF.7) At the beginning of the Results part the authors should specify in which system they are working.8) The green line is not mentioned in the legend to Met cosedimentation”.9) A-site/E-site allostery has been recently reported by Ferguson et al., 2015, Mol Cell for human 80S ribosomes. This is relevant for the respective discussion in the third paragraph of the subsection “Rates of oligopeptide formation and Met-tRNAeLife. Your revised article has been favorably evaluated by John Kuriyan (Senior editor), a Reviewing editor, and two reviewers.Thank you for resubmitting your work entitled "Kinetics of initiating polypeptide elongation in an IRES-dependent system" for further consideration at The manuscript has been improved but there are some remaining issues raised by Reviewer #3 that need to be addressed before acceptance. In considering these new comments, Reviewer #2 also asks that you carefully review the Materials and methods to insure that they contain all of the critical information that would be required to duplicate the work.Reviewer #2:eLife.The authors have significantly improved the manuscript by satisfactorily addressing the comments and concerns of the reviewers. The story they prevent extends our knowledge on the function IRES-dependent systems and can be published in Reviewer #3:In the revised version Zhang and colleagues have addressed many points raised by the reviewers and have improved their paper. However, there are still some points where discussion should be extended.1) The authors now confirm that the effect of eEF2 is to inhibit k-1 and write that this is "consistent with its role as a translocase". However, the accepted role of EF-G/eEF2 is to accelerate translocation, not to inhibit back-translocation. This warrants additional discussion.2) The authors have pre-incubated 80S-FVKM-IRES complexes with eEF2-GTP for 1 – 2 hr. How they can rule out that GTP consumption has an impact on the experiment? Is it possible that accumulation of eEF2-GDP during pre-incubation results in a significant fraction of 80S FVKM-IRES-eEF2 complexes (between Structures 1 and 2) to slow down k2.3) The authors write that the anisotropy assay in Phe is labile. Can they estimate the off-rate k-2. Is it valid to neglect k-2 for numerical integration?4) The authors state that A-site-bound Phe-tRNA Essential revisions:The following issues raised in the referees' comments shown below need to be addressed in the revised manuscript.Ref. #1: All of the requests for clarification of reaction conditions or interpretations of results should be addressed with appropriate revisions of text.This has been addressed.Ref.#2: The request for complete details about fitting the kinetic data in comment #1 should be honored.This has been addressed.As described in comment #2, the need for modifying the IRES sequence should be more explicitly stated in Materials and methods, as it would have been technically feasible to carry out all experiments with the WT sequence.Phe was available to us and the tRNAAla acceptor was not, and Eric Jan told us that the Phe codon UUC could be substituted for the Ala GCU codon with little or no functional consequence.This was simply a matter of convenience, as now stated explicitly in the first paragraph of the Materials and methods. When we started this work tRNAIn addition, it is important to compare the rates of IRES-mediated translation given by the native sequence versus your modified IRES sequence either in a cell-free extract or in your reconstituted system.This has been addressed by addition of To respond to points 3. & 4., it would be sufficient to revise the text to remove statements about allostery; for point 5, to explain why you monitored the puromycin reaction as you did in These points have been addressed, but, as explained in response to Reviewer #2, point #3, we would prefer to leave the allosteric discussion in the text.1/2 of step 1. You must also discuss the discrepancy with the previous results, particularly those of Ruehle et al.Ref. #3: Points 1.-3 concern your conclusion that eEF2 does not stimulate the rate of TC binding in step 2 and rather only shifts the equilibrium in step 1 towards structure 2, which is at odds with other published work. As this is a hypothetical interpretation of the biphasic kinetics in 1/2 values for Steps 1 and 2 in revised We disagree with the request for a toeprinting experiment, for reasons elaborated in response to Reviewer 3’s comments #s 1 – 3. However, we have carried out a more complete analysis of our kinetic data, resulting in the addition of a new table revised  and a moYou should clarify the issues raised in points 4-6 with the appropriate revisions of text.We have addressed points 4 and 6 in our response. We disagree with point 5, as stated in our detailed response.Original reviews:Reviewer #1:The results are significant in providing strong evidence that the two pseudotranslocation steps , as well as peptidyl-Met-tRNAMet binding to the P-site (Structures 10 and 13), efficiently…", the clarity could be improved by mentioning the specific peptidyl-tRNAs by their amino acid compositions for each of the structures, as "peptidyl-Met-tRNAMet" is imprecise nomenclature.1) In the second paragraph of the subsection “Rates of oligopeptide formation and Met-tRNAThe requested clarifications have been made, both in the Results and Experimental Sections.Met cosedimentation”: Also, it's not perfectly clear whether the instability of Met-tRNA binding to the A-site observed for structure 3 and 5, and FM-tRNA to structure 6 using the co-sedimentation assay is unusual; whereas stable binding of both Met-tRNA and peptidyl-tRNA to the A-site in structures 8, 9, 11-12 is typical of A-site-bound ligands in conventional (non-IRES) elongation complexes; and this distinction needs to be carefully spelled out.2) In the subsection “Rates of oligopeptide formation and Met-tRNAMet cosedimentation”, end of second paragraph and start of the third paragraph.The requested distinction has been made. See subsection “Rates of oligopeptide formation and Met-tRNA3) In the second paragraph of the subsection “Translocation of tetrapeptidyl-tRNA (Step 12) is much more rapid than of tripeptidyl-tRNA (Step 9)”: requires citation of a figure for puromycin synthesis rates.Lys, demonstrating that A-site reactivity is approximately 20-fold less than P-site reactivity. This result, which has been added to Lys is much slower than translocation of PheValLysMet-tRNAMet. We also added two sentences (in the aforementioned paragraph) explicitly comparing A-site vs. P-site reactivity with puromycin in prokaryotic and eukaryotic ribosomes.The references for the reactivity of P-site bound aminoacyl-tRNA with puromycin in eukaryotic ribosomes were originally provided . In writing the revised version we became aware of a paper published in late 2015 by Ferguson et al., which also provided a pertinent result, and this reference was also added. In addition, in considering this comment we realized that there were no reliable data extant for the reactivity of A-site bound aminoacyl-tRNA with puromycin in eukaryotic ribosomes. Accordingly, we performed the relevant experiment for A-site bound PheValLys-tRNA4) In the second paragraph of the subsection “Translocation of tetrapeptidyl-tRNA (Step 12) is much more rapid than of tripeptidyl-tRNA (Step 9)” and More detail was added to the description of the puromycin reaction in the subsection entitled “Rates of puromycin adduct formation” as requested. In addition, the reference to 5) In the last paragraph of Results: more detail is required to understand how binding of peptidyl tRNA harboring labeled Met is distinguished from Met-tRNA binding to the A-site in these assays.Met has been added to the subsection entitled “Octapeptide” in the Experimental section. We also added A sentence justifying the use of the cosedimentation assay to measure octapeptide formation involving peptide transfer to ribosome-bound Met-tRNA6) Error estimates have not been defined in any of the figures or tables and this needs to be rectified.1/2 values for the combined steps 4-8 and 7-11.In the subsection labeled “Kinetic measurements” the last sentence has been amended to read “Error bars in figures are shown as average deviations.” In addition, several additions were made to revised Reviewer #2:The paper by Cooperman et al. provides a first kinetic description of the initial steps of translation by eukaryotic ribosomes after the initiation at an IRES. There are very few papers on mechanistic aspects of eukaryotic translation in general and the present work provides very interesting, significant insights. It is a real challenge to obtain clean kinetic data for the eukaryotic system and this paper provides valuable information on the kinetic properties of eukaryotic ribosomes. The technical quality of the data is high  and the conclusions are warranted. My only reservation is that many technical aspects are not clearly described. Given that this paper delineates new kinetic approaches to study eukaryotic translation, it is particularly important to provide a very detailed description of methods. This really has to be carefully revised. I personally do not like the title, because it is not informative at all; a more rigorous title would be better.The title has been changed as requested.Detailed comments:1) Fitting of the kinetic data in this paper is mostly non-exponential and would require fitting to a model by numerical integration. This part is completely missing in Materials and methods. This must be described in all details required to understand and reproduce the calculations.Only the results presented in 35S]Met; in this case one could use the native sequence. The need for modifying the sequence should be more explicitly stated in Materials and methods. Clearly, the whole set of experiments cannot be repeated with Ala as a 1st amino acid; however, it would be highly desirable to compare IRES-mediated translation with a native sequence and a modified sequence in the cell-free translation system .2) Among all potential criticism of the experimental system , I am particularly worried about the altered coding sequence. It is not clear why the native Ala codon was replaced by Phe, as unlabeled amino acids are used anyway and the tRNAs are prepared by a method which should readily yield tRNA(Ala), i.e. one could label tRNA(Ala) with Prf . This means that in principle one could use the native sequence. Furthermore, the measurements and rate determinations would be even more straightforward if [14C] and [3H]-labeled amino acids were used instead of Met (at least to some extent) even in the absence of the mRNA. Are there controls for that?7) In the subsection “Complex preparations. TCs and various 80S·IRES complexes”. 30S subunits were added to the centrifugation assays as a carrier. However, 30S subunits can bind [Yes. Co-sedimentation due to binding to 30S subunits is negligible. This point has been addressed by adding both an appropriate sentence to the subsection in Materials and methods entitled “Complex Preparations”and 8) The reviewer is referring to the 35S]Met-TC”. My expectation is that in the absence of the preceding Lys-TC Met should not be incorporated at all. This has to be clarified.9) Clarifying text has been added. The basic point is that, in 4C, the kinetics of tripeptide synthesis was followed for either 5 steps (Structure 4 converted to Structure 9) or two steps (Structure 7 converted to Structure 9). Similarly, in 4D the kinetics of tetrapeptide synthesis was followed for either 5 steps (Structure 7 converted to Structure 12) or two steps (Structure 10 converted to Structure 12).10) In the subsection “Kinetic measurements”, "averages of 2-4 independent determinations" – do the authors mean technical replicates or independent experiments ?Clarifying language on this point has been added to the subsection “Kinetic measurements”.Reviewer #3:Zhang and colleagues have investigated the effect of the CrPV IRES RNA on the first rounds of translation elongation including the initial pseudo-translocation steps. They have performed for the first time experiments to determine the rates of the various main steps of the elongation cycles. They show that the IRES initial significantly slows down the translocation steps are down until the tetrapeptide stage is reached. The CrPV is an important minimal model system for translation initiation in eukaryotes The results are novel and interesting. However, the taking into account the following points is recommended before the paper can be published.1) According to The reviewer makes two suggestions for revising the manuscript in this point, and we disagree with both.There is no conflict with the Ruehle et al. paper, on which we are co-authors. The colocalization result is reported in Figure 6 of Ruehle et al. and utilizes single molecule observation. The relevant experimental section describing this experiment in the Ruehle et al. paper reads as follows:“The 80S-IRES ribosome complex was incubated in the flowcell for 5 min and components that remained untethered to the surface of the microfluidic flowcell at the conclusion of the 5 min were washed out of the flowcell using an imaging buffer…”.Phe, accounting for the much lower stoichiometry observed in the absence of added eEF2. This point is addressed directly in Ruehle et al. in the section entitled “Loop 3 facilitates eEF2’s ability to translocate ac-tRNA on IGR IRES-80S ribosome complexes”:This washing procedure has the effect of removing the labile A-site bound Phe-tRNA“To examine eEF1A-dependent ac-tRNA delivery, we assembled TC with Phe353Phe(Cy5)+eEF1A+GTP and delivered this to the immobilized IRES-80S complexes without eEF2. Compared to the reactions lacking eEF1A, both IRESs show increased and similar ac-tRNA occupancies . These data initially seem at odds with the anisotropy data in which eEF2-independent ac-tRNA association with 80S-WT IRES ribosome complexes is much greater than complexes with Δ3. This apparent discrepancy is likely due to the fact that anisotropy data are obtained under equilibrium conditions where transient interactions are observed, whereas the single-molecule fluorescence data are collected after the flowcell is flushed and thus only show stable long-lived association.”tRNAThe second suggestion requests that we clarify “the nature of the complex” through use of a toeprinting experiment. We are not completely clear to which complex the Reviewer is referring: Structure 2 with the A-site empty in either the absence or presence of eEF2; or Structure 3, with aa-tRNA bound in a labile manner in the A-site. Of these three possibilities, we consider two to be noncontroversial. In the absence of eEF2 and Phe-TC, our results suggest that the equilibrium between Structures 1 and 2 strongly favors 1, in agreementwith the results of others. With respect to Structure 3, the results presented in the Ruehle et al. paper show the tRNA binding to be enhanced for cognate vs. non-cognate tRNA, supporting the structure shown.Where our results may appear to be at odds with published results, likely prompting the Reviewer’s suggestion, are for Structure 2 in the presence of added eEF2·GTP. We address this point directly in the revised text . The basic conclusion is that the reaction conditions employed in the toeprinting assay make it an unreliable method for measuring eEF2·GTP effects on the 1 to 2equilibrium position, in agreement with an earlier suggestion of Muhs et al. (2015) , which has recently been supported by results of Petrov et al. (2016). Because of this, we do not agree with the reviewer that a toeprinting experiment is likely “to establish the nature of the complex in a more direct manner”.2) The authors state that k-1 is much smaller than k2 and was assumed to be negligible. How has this been determined? In fact, a toeprint signal indicative of translocation cannot be obtained by incubation of binary 80S-IRES complexes with eEF2 alone, but requires an A-site ligand, too . This suggest that the translocated complex (structure 2) is kinetically labile and contradicts the present statement that the back-translocation is negligible. Again, the authors should do experiments, e.g. toeprinting, to provide additional evidence that pre-incubation with eEF2-GTP shifts the equilibrium between structures 1 and 2 from 95:5 to 50:50 as stated.1/2 of step 1. How this is possible? Do the authors propose that k-1 is becoming smaller, i.e. that eEF2 slows down back-translocation without accelerating forward translocation?3) The explanation about the effect of eEF2 on the equilibrium between structure 1 and 2 (Discussion) is curious. They propose that eEF2 shifts the equilibrium towards structure 2 at the same time do not measure a significant effect on the tk-1 >> k1, whereas in the presence of eEF2·GTP, k-1 ~ k1. This change results from the effects of eEF2·GTP in strongly decreasing k-1 (~ 50-fold) while only modestly decreasing k1 (~ 2-fold). The rate constants in 1/2 values for Steps 1 and 2 in These two valid points are linked, and we thank the reviewer for raising them. To address them we fit the anisotropy results presented in 4) Can the authors rule out that back-translocation or peptidyl-tRNA drop off influences the following steps towards tetrapeptide synthesis?We do not think that peptidyl-tRNA drop off is a major issue, because the yield of peptide per ribosome is constant for di-, tri- and tetrapeptide. However, we acknowledge that many of the details of the process, including possible back translocation during oligopeptide synthesis, remain to be elucidated.Additional points:5) The first sentence of the paper "Initiation of protein synthesis in eukaryotic cells proceeds via two well-established pathways" is to some extent misleading. As the authors acknowledge later factor requirement of different IRES RNAs is diverse and the structure and mechanism of different IRES RNAs is also different. So there are several pathways leading to internal initiation.We disagree with the reviewer. Our division of initiation of protein synthesis via two pathways is a common way of introducing the subject and follows the presentation in a review article we cite . The qualification that IRES-initiated synthesis can take place in diverse ways comes three lines later in the opening paragraph, so we are hardly neglecting this point.6) The peptide coding sequence is not really part of the IRES. Therefore, it is misleading to talk about mutant IRES, when the changes are in the ORF.We accept this criticism and have made the appropriate changes in the Results section and in 7) At the beginning of the Results part the authors should specify in which system they are working.The requested change has been made in revising the first sentence of Results.8) The green line is not mentioned in the legend to This criticism is addressed in the revised last sentence of the Met cosedimentation”.9) A-site/E-site allostery has been recently reported by Ferguson et al., 2015, Mol Cell for human 80S ribosomes. This is relevant for the respective discussion in the third paragraph of the subsection “Rates of oligopeptide formation and Met-tRNAeLife. It is relevant to our manuscript with respect both to A-site:E-site allostery  and puromycin reactivity. The manuscript has been altered to include references to Ferguson et al. (2015) in the subsections: “Rates of oligopeptide formation and Met-tRNAMet cosedimentation”, last paragraph; “Translocation of tetrapeptidyl-tRNA (Step 12) is much more rapid than of tripeptidyl-tRNA (Step 9), third paragraph and Discussion, fourth paragraph.We agree. We only became aware of the Ferguson et al. article (published November 2015) after we had submitted our initial MS to The manuscript has been improved but there are some remaining issues raised by Reviewer #3 that need to be addressed before acceptance. In considering these new comments, Reviewer #2 also asks that you carefully review the Materials and methods to insure that they contain all of the critical information that would be required to duplicate the work.Reviewer 2 raises no specific objections to the previous submission (Revision 1) but we note that some experimental detail has been added to the Reviewer #3:In the revised version Zhang and colleagues have addressed many points raised by the reviewers and have improved their paper. However, there are still some points where discussion should be extended.1) The authors now confirm that the effect of eEF2 is to inhibit k-1 and write that this is "consistent with its role as a translocase". However, the accepted role of EF-G/eEF2 is to accelerate translocation, not to inhibit back-translocation. This warrants additional discussion.We strongly disagree with the reviewer on this point. In fact, a principal role of EF-G, the prokaryotic equivalent of eEF2, has long been considered to be to inhibit back-translocation , a view backed up by recent experiments. A reference to a particularly compelling set of results  has been added to the text to make this point explicit .2) The authors have pre-incubated 80S-FVKM-IRES complexes with eEF2-GTP for 1 – 2 hr. How they can rule out that GTP consumption has an impact on the experiment? Is it possible that accumulation of eEF2-GDP during pre-incubation results in a significant fraction of 80S FVKM-IRES-eEF2 complexes (between Structures 1 and 2) to slow down k2.oC in buffer 4 supplemented with 1 mM GTP”. To eliminate any ambiguity, we have made two additions to the We understand the reviewer’s concern, and have added additional text to make it clearer that we have adequately addressed this concern. As stated in the experimental section (subsection “Kinetic Experiments”) “Unless otherwise noted, all reactions were performed at 37 3) The authors write that the anisotropy assay in The reviewer is correct, but as Phe-TC is bound to the 80S·IRES complex in both Structures 3 and 4, any anisotropy change accompanying Structure 4 formation from Structure 3 is expected to be minor.Phe is labile. Can they estimate the off-rate k-2. Is it valid to neglect k-2 for numerical integration?4) The authors state that A-site-bound Phe-tRNAPhe would not be measured by k-2, but by a separate reaction following dissociation of eEF1A·GDP from the ribosome, the kinetics of which we have not measured. However, this question by the reviewer prompted us to add a second sentence to the -2, which describes the reversible binding of ternary complex, the answer is that it is always valid to use the minimum number of parameters to fit the available data. This results in obtaining apparent kinetic constants, as listed in -2 to be estimated, with, perhaps, altered apparent values of k1, k-1, and k2.Productive TC binding is accompanied by GTP hydrolysis, an essentially irreversible step. Thus, the lability of bound Phe-tRNA


YB-1 mRNA translation was caused by suppression of activity of the mTOR signaling pathway. It was suggested that this event may be mediated by phosphorylation of the 4E-binding protein (4E-BP). Here, we report that 4E-BP alone can only slightly inhibit YB-1 synthesis both in the cell and in vitro, although it essentially decreases binding of the 4F-group translation initiation factors to mRNA. With inhibited mTOR kinase, the level of mRNA binding to the eIF4F-group factors was decreased, while that to 4E-BP1 was increased, as was observed for both mTOR kinase-sensitive mRNAs and those showing low sensitivity. This suggests that selective inhibition of translation of YB-1 mRNA, and probably some other mRNAs as well, by mTOR kinase inhibitors is not mediated by the action of the 4E-binding protein upon functions of the 4F-group translation initiation factors.The Y-box binding protein 1 (YB-1) is a key regulator of gene expression at the level of both translation and transcription. The mode of its action on cellular events depends on its subcellular distribution and the amount in the cell. So far, the regulatory mechanisms of YB-1 synthesis have not been adequately studied. Our previous finding was that selective inhibition of  YB-1 is a multifunctional DNA- and RNA-binding protein belonging to the family of proteins with an evolutionary conserved cold shock domain. YB-1 participates in many cellular events and is involved in virtually all stages of transfer and realization of the genetic information, specifically, transcription and mRNA translation1YB-1 mRNA translation to activity of the mTOR signaling pathway that regulates cell growth and cell response to various growth stimuli, stresses, and availability of nutrients and energy56As we showed previously, YB-1 synthesis depends on the rate of cell division. Suppression of cell division resulted in selective inhibition of YB-1 synthesis in the cell, while its recovery caused a rapid and specific increase in YB-1 synthesisYB-1 mRNA. However, rapamycin (another mTOR inhibitor affecting mostly p70S6 kinase) produced no effect on YB-1 synthesisIt was our previous finding that mTOR inhibition by selective inhibitors pp242 and Torin2, that suppress phosphorylation of 4E-BP and p70S6 kinase, caused in the cell specific inhibition of both YB-1 synthesis and translation of reporter mRNAs carrying the 5′ UTR of YB-1 mRNA translation under mTOR inhibition. We showed that overexpression of mutant non-phosphorylatable 4E-BP1 in the cell or addition of recombinant 4E-BP1 to a cell-free translation system produced no noticeable effect either on synthesis of endogenous YB-1 in HeLa cells or translation of reporter mRNA carrying the YB-1 mRNA 5′ UTR. Next, affinity chromatography on resin with immobilized capped mRNA fragments was used to show that, upon mTOR kinase inhibition, fragments of both mTOR sensitive and nonsensitive mRNAs demonstrated equally decreased binding to the factors eIF4E, eIF4G, and particularly eIF4A, with increasing binding to 4E-BP. Supposedly, specific inhibition of translation of YB-1 mRNA and some other mRNAs occurring concurrently with inhibition of the mTOR signaling pathway is not explained by specifically changed affinity of the translation initiation factors for these mRNAs. Besides, according to our data, in the course of translation suppression with mTOR inhibitors, the crucial event is not only 4E-BP dephosphorylation preventing eIF4G-to-eIF4E binding on the mRNA but probably a lower level of mRNA binding to eIF4E and eIF4A, as well. This suggested a higher sensitivity of translation of YB-1 mRNA and similar mRNAs to inhibition of cap-binding and RNA-helicase activities, which has been confirmed experimentally.The current study is aimed to learn whether or not 4E-BP is the key protein in regulation of YB-1- and eEF1A mRNAs was strongly suppressed (~10-fold), in contrast to control mTOR-nonsensitive mRNAs with 5′ UTRs from β-glob-, Apaf1- and GADD45 mRNAs (~1.5–2-fold)  . It shoutrol see . Like antrol see . Therefotrol see  manifestrps20 mRNA 5′ UTR was expected to be also strongly suppressed as a result of mTOR inhibitionrps20 mRNA because, as believed, TOP mRNAs (and rps20 mRNA among them) require a terminal position of the TOP sequence at the 5′ end. However, it should be noted that the reporter mRNA carrying 5′ UTR of another TOP mRNA, namely, eEF1A mRNA, retained the degree of its sensitivity to mTOR inhibition. However, it cannot be ruled out that this degree would be below the one observed in the case of natural eEF1A mRNA 5′ UTR.Translation of mRNAs carrying the YB-1 mRNA. As seen from YB-1- and eEF1A mRNAs, as compared to mTOR-nonsensitive mRNAs used as a control. The inhibitory effect was insufficient, and the translation level accounted for 70% of the initial value, as observed in contrast to mTOR inhibition-induced suppression of translation of mTOR-sensitive reporter mRNAs in HeLa cells.According to the literature, mRNA sensitivity for mTOR inhibition is 4E-BP-dependentluciferase mRNA containing 5′ UTR from β-globin mRNA . Nevertheless, translation of this mRNA was not at all inhibited. Probably, the reason for the minor effect of 4E-BP1 was that the initially high proportion of non-phosphorylated 4E-BP in Krebs extract could hamper further inhibition ) overexpressed in HeLa cells  failed tB-1 mRNA  or inhibB-1 mRNA .luciferase mRNA carrying YB-1 mRNA 5′ UTR was biotinylated, capped, and incubated with control HeLa cell lysate or lysate of 4E-BP1(4Ala)-overexpressing HeLa cells using the same buffer as for the cell-free translation system. After immobilization of biotinylated RNA on Streptavidin-Sepharose, RNA-bound proteins were eluted and analyzed by Western blotting to detect translation initiation factors 4E, 4G, 4A, and 4E-BP1. It is seen that 4E-BP1(4Ala) effectively binds to the capped biotinylated reporter mRNA carrying YB-1 mRNA 5′ UTR . In experiment, A 5′ UTR . This biA 5′ UTR .YB-1 mRNA translation is mediated solely by the 4E-binding protein. Probably, apart from the presence of hypophosphorylated 4E-BP1, efficient inhibition of translation requires an additional mTOR inhibition-related event.So, it is doubtful whether under mTOR inhibition the suppression of YB-1 mRNA translation inhibition under the effect of mTOR kinase inhibition, we monitored binding of translation initiation factors to the 5′-terminal sequence of reporter luciferase mRNA carrying 5′ UTRs of various mRNAs ; the effect of the last two treatments is believed to be mediated mostly by the mTOR signaling pathway9To elucidate the mechanism of specific us mRNAs . In expeYB-1 mRNA and β-glob mRNA demonstrated the same changes as in the case of mTOR inhibition with Torin2. However, the absence of eIF4A and eIF4G binding observed here can be explained not by their lessened affinity for RNA but by a considerable decrease in their amount after 16 h hypoxia  and those with translation almost independent of mTOR inhibition . This suggests that specific inhibition of translation of YB-1 mRNA and some other mRNAs observed upon inhibition of the mTOR signaling pathway resulted not from specifically altered affinity of initiation factors but probably from a stronger dependence of YB-1 mRNA translation on the eIF4F-group factors. However, since in the absence of inhibition of reporter mRNA translation the overexpression of non-phosphorylatable 4E-BP1 led to decreased binding of the eIF4F-group initiation factors, it is of importance that Torin2-induced mTOR inhibition caused not only inactivation of the eIF4F-group factors but also that of other proteins responsible for cap-binding and RNA-helicase activities during the cap-dependent translation.It was of particular importance to us that binding modulations of the eIF4F-group factors and 4E-BP1 appeared to be the same both for mRNAs with translation suppressed upon mTOR inhibition (7GpppG), and in particular, helicase 4A inhibitors hippuristanol and dominant-negative eIF4A mutant R362Q (eIF4A (R362Q)). Hippuristanol is a small molecule which mostly modulate the eIF4A activityIt was hypothesized that mTOR inhibition has a more dramatic effect on mRNAs whose translation shows a stronger dependence on initiation factors mediating the cap-dependent translational mechanism. To verify this supposition, we checked up whether translation of the used reporter mRNAs in Krebs extracts was affected by the following inhibitors of cap-dependent translation: the cap-analog  or by hippuristanol showed similar results, except for 5′ UTR from bin mRNA . Close rbin mRNA . Importabin mRNA . Thus, oApaf1 mRNA 5′ UTR resistant to mTOR kinase inhibitors displayed high sensitivity to both addition of eIF4A (R362Q) and hippuristanol to Krebs extract and addition of hippuristanol to HeLa cells. This is hardly surprising, because, as shown, this mRNA has a long structured 5′ UTR and is translated by the 5′-end-dependent scanning mechanismluciferase mRNA carrying the 5′ UTR from Apaf1 mRNA was also inhibited by the cap-analog, though to a lower degree than in the case of luciferase mRNA carrying the YB-1 mRNA 5′ UTR. However, in spite of a decreased level of eIF4A and eIF4E binding to Apaf1 mRNA 5′ UTR  is insufficient for potent suppression of mRNA translation; presumably, mTOR inhibitors additionally influence the translation initiation mechanisms, thereby promoting specific inhibition of some mRNAs.Meanwhile, translation of Supposedly, this additional effect occurring upon mTOR inhibition consists in 4E-BP-independent inhibition of the cap-binding and RNA-helicase activities of factors involved in translation initiation, including the canonical initiation factors eIF4E and eIF4A. The role of 4A is not a novel finding, since the protein PDCD4, a substrate of this pathway, is shown to act as an inhibitor of eIF4A activity96YB-1 mRNA, we showed that its translation is only slightly resistant to inhibition of activity of translation initiation factors by the cap-analog (mutant eIF4A (R362Q)) and by hippuristanol. The reason for such a behavior is unclear, and it possibly differs from mRNA to mRNA or from one small group of mRNAs to another; it may result from peculiarities of the secondary structure or binding of certain proteins to a certain sequence within the 5′ UTR, or something else. Probably this explains the diversity of proteins interacting with the TOP-motif  and allegedly participating in translation regulation8Our supposition is that specific inhibition of mRNA translation upon suppression of the mTOR signaling pathway is underlain not by specifically altered affinity of initiation factors but probably by a stronger dependence of mRNA translation on activity of the eIF4F-group factors or other proteins of similar activities. Specifically for YB-1 mRNA may be classified with mRNAs sensitive to both cap-binding and RNA-helicase activities.Besides, it seems most probable that among mRNAs sensitive to mTOR inhibition there are those highly sensitive to suppression of the eIF4E activity  or to inhibition of eIF4A activity, and those depending on both eIF4E- and eIF4A activities. This categorization may explain a distinct division of all mRNAs into two groups: those with translation suppressed by the mTOR inhibitor INK128 and those with translation inhibited by silvestrol, an inhibitor of the eIF4A activityApaf1 mRNA 5′ UTR are of significance. This mRNA is extremely sensitive to eIF4A activity inhibition both in vitro and in vivo, but its translation remains unsuppressed by mTOR inhibition implying a decreased level of mRNA-to-eIF4A binding. This may be explained by the fact that the first step in mTOR inhibition is shutdown of the canonical scanning mechanism involving eIF4A, probably without inhibition of other RNA helicases that replace eIF4A in Apaf1 mRNA 5′ UTR scanning. Together with a somewhat lower sensitivity of Apaf1 mRNA to the decreasing amount of accessible eIF4E, this replacement allows translation in conditions of inhibited mTOR. However, in the case of incubation of these cells with hippuristanol, that presumably inhibited not only 4A but also other helicases required for scanning the Apaf1 mRNA 5′ UTR, translation of this mRNA was suppressed. The fact that in Krebs extract translation of mRNA with the Apaf1 mRNA 5′ UTR was inhibited by addition of eIF4A (R362Q) may be explained by the dual activity of this mutant: it supported affinity for eIF4G but prevented interaction of eIF4G with other helicases. Also, it is worth noting a significant decrease of 4E-BP1 binding to Apaf1 5′ UTR, as compared with β-globin and YB-1 UTRs  and Dhh1/RCK/DDX6Interestingly, the helicase eIF4A appeared to be the only protein with a lower level of binding to capped mRNA under serum (growth factors) starvation conditions. This not only points to the importance of the eIF4А activity in regulation of translation with deficient growth factors but also emphasizes the difference between mTOR inhibitor-involving and uninvolving mechanisms of translation regulation. Lately, this hypothesis gains more and more evidence-globin-FLuc-3′ UTR GAPDH-A50 were obtained as described previouslyThe pSP36T-5′ UTR YB1-FLuc-3′ UTR GAPDH-A50 and pSP36T-5′ UTR βAAGCTTAAGAAGAGGTAGCGAGTGGACGT-3′, the reverse primer was 5′-ATCCATGGTCCCTCAGATCTTTCTC-3′ (the HindIII and NcoI restriction sites are bold). The forward primer for rps20 5′ UTR was 5′-TGAAGCTTCCTTTCTTTTTGAGGAAGAC-3′, the reverse primer was 5′-TGCCATGGCTGTTGCGCGCGGGCTT-3′ (the HindIII and NcoI restriction sites are bold). The PCR products were treated with HindIII and NcoI and ligated into pSP36T-5′ UTR β-globin-FLuc-3′ UTR GAPDH-A50 treated with the same restriction endonucleases. The constructs were named pSP36T-5′ UTR Apaf1-FLuc-3′ UTR GAPDH-A50 and pSP36T-5′ UTR rps20-FLuc-3′ UTR GAPDH-A50.The 5′ UTRs of Apaf1 and rps20 mRNAs were obtained by RT-PCR amplification of total RNA of HeLa cells. The forward primer for Apaf1 5′ UTR was 5′-ATAGCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTGTCGTGAAAACTACCCCTAAAAGCCAAAC-3′ and 5′-CATGGTTTGGCTTTTAGGGGTAGTTTTCACGACACCTGTGTTCTGGCGGCAAACCCGTTGCGAAAA-3′; sticky ends similar to those formed after HindIII and NcoI restriction are shown in bold) followed by their ligation into pSP36T-5′ UTR β-globin-FLuc-3′ UTR GAPDHA50 treated with NcoI and HindIII restriction endonucleases. The construct was named pSP36T-5′ UTR EEF1A-FLuc-3′ UTR GAPDH-A50.The 5′ UTR of EEF1A mRNA was obtained by annealing two oligonucleotides (5′-pGL3–5′ UTR GADD45-Luc was kindly provided by Dr. Ilya Terenin.RLuc RNA was obtained by PCR amplification of the pGL3-RLuc plasmidDNA template for DNA templates for RNA fragments used in protein purification experiments were obtained by PCR amplification of the corresponding plasmid with the SP6 promoter forward primer 5′-ATTTAGGTGACACTATAG-3′ and the reverse primer 5′-CCAGGGCGTATCTCTTCATAGCCT-3′ complementary to the sequence of luciferase cDNA (83–106 nt).Firefly Luciferase (FLuc) cap−poly(A)+ mRNA with YB-1 mRNA 5′ UTR and GAPDH mRNA 3′ UTR was transcribed by SP6 RNA polymerase from pSP36T-5′ UTR YB1-FLuc-3′ UTR GAPDH-A50 linearized with HpaI. Firefly Luciferase (FLuc) cap−poly(A)+ mRNA with β-globin mRNA 5′ UTR and GAPDH mRNA 3′ UTR was transcribed by SP6 RNA polymerase from pSP36T-5′ UTR β-globin-FLuc-3′ UTR GAPDH-A50 linearized with HpaI. Firefly Luciferase (FLuc) cap−poly(A)+ mRNA with Apaf1 mRNA 5′ UTR and GAPDH mRNA 3′ UTR was transcribed by SP6 RNA polymerase from pSP36T-5′ UTR Apaf1-FLuc-3′ UTR GAPDH-A50 linearized with HpaI. Firefly Luciferase (FLuc) cap−poly(A)+ mRNA with EEF1A mRNA 5′ UTR and GAPDH mRNA 3′ UTR was transcribed by SP6 RNA polymerase from pSP36T-5′ UTR EEF1A-FLuc-3′ UTR GAPDH-A50 linearized with HpaI. Firefly Luciferase (FLuc) cap−poly(A)+ mRNA with rps20 mRNA 5′ UTR and GAPDH mRNA 3′ UTR was transcribed by SP6 RNA polymerase from pSP36T-5′ UTR rps20-FLuc-3′ UTR GAPDH-A50 linearized with HpaI. These reporter mRNAs had the same additional 12 nt sequence upstream of 5′ UTR (see  UTR see .Firefly Luciferase (FLuc) cap−poly(A)+ mRNA with GADD45 mRNA 5′ UTR was transcribed by T7 RNA polymerase from pGL3–5′ UTR GADD45-Luc linearized with XbaI.Renilla luciferase (RLuc) mRNA was transcribed by T7 RNA polymerase from corresponding PCR product.luciferase mRNA coding region.RNA fragments used in protein purification experiments were transcribed by SP6 RNA polymerase from corresponding PCR products and contained 5′ UTRs of various mRNAs and a 106 nt sequence of the 7G Capping System and ScriptCap 2′-O-Methyltransferase Enzyme (CellScript) according to the suppliers’ recommendations.Transcription was performed as described previously2. Cells were passaged by standard methods.HeLa cells were cultivated in DMEM/F12 supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 μg streptomycin (PanEco). Cells were kept at 37 °C in a humidified atmosphere containing 5% CO2 by N2 displacement, 5% CO2) in a multigas incubator  for 16 h. After treatment cells were washed with phosphate buffered saline and lysed in hypotonic buffer .Сells were treated with Torin2 (Tocris), or Hippuristanol  for 1 h before RNA transfection. To prepare lysates for affinity chromatography using mRNA fragments immobilized on Streptavidin-Sepharose, HeLa cells (two 100-mm dishes per experiment) were cultivated either with Torin2 (250 nM) for 3 h or with Hippuristanol (2 and 4 μM) for 3 h, or in DMEM/F12 without fetal bovine serum for 48 h, or under hypoxia . For a typical RNA transfection, 0.5 μg of FLuc mRNA and 0.05 μg of RLuc mRNA was incubated with 2 μl of the transfection reagent in 100 μl DMEM for 20 min and then added to the growth medium. 2 h later cells were harvested, and luciferase activities were analyzed using a Dual Glo Luciferase Assay kit (Promega). All transfections were repeated several times in different cell passages.35S]-methionine labeling experiment.For DNA transfection experiments, HeLa cells were plated onto a 24-well dish 24 h prior to transfection of DNA. Cells were transfected using Lipofectamine 2000 (Invitrogen). For transfection, 1 or 2 μg of the appropriate plasmid  for 3 h. These cells were utilized for immunoprecipitation.For [2, 2 units of Human Placental Ribonuclease Inhibitor (Fermentas), 0.15 pmol FLuc mRNA, and 0.015 pmol RLuc mRNA for 1 h. The luciferase activities were measured using a Dual Luciferase Assay kit. When indicated, various amounts of m7GpppG  were added, and the translation mix was pre-incubated for 5 min before addition of mRNA. For experiments with 4E-BP1, the recombinant protein was used. Plasmid pGEX-6p1-h4E-BP1  encoding human 4E-BP1-GST fusion was expressed in Escherichia coli, and 4E-BP1 was purified using Glutathione-Sepharose 4B and PreScission Protease (Amersham) and dialyzed against buffer A . Purified 4E-BP1 was added to the translation mix in a volume less than 1.5 μl and incubated for 5 min before addition of reporter mRNAs. In control, an equal amount of buffer A was added.Translation experiments were performed in a total volume of 10 μl which contained 5 μl of the Krebs (ascites carcinoma cells) S30 extract (kindly provided by Prof. Ivan Shatsky’s team), 1 μl 10X translation buffer , 100 mM KAc, 1 mM Mg(Ac)Escherichia coli, and eIF4A(R362Q) was purified using TALON® Metal Affinity Resin (Clontech) and dialyzed against buffer A . Purified eIF4A (R362Q) was added to the translation mix in a volume less than 1.5 μl and incubated for 5 min before addition of reporter mRNAs. In control, an equal amount of buffer A was added.For experiments with eIF4A(R362Q), the recombinant protein was used. Plasmid pET(His6-R362Q)S2448 , polyclonal rabbit antibody against mTOR , polyclonal rabbit antibody against phospho-AKTS473 , polyclonal rabbit antibody against AKT , monoclonal rabbit antibody against phospho-p70S6KT389 , monoclonal rabbit antibody against phospho-rpS6S240/244 , polyclonal rabbit antibody against 4E-BP1 , monoclonal rabbit antibody against phospho-4E-BP1T37/46 , monoclonal rabbit antibody against nonphospho-4E-BP1 , monoclonal rabbit antibody against eIF4E  polyclonal rabbit antibody against phospho-eIF4ES209 , monoclonal rabbit antibody against phospho-eIF2αS51 , monoclonal rabbit antibody against eIF4G , polyclonal rabbit antibody against phospho-eIF4GS1108 , polyclonal rabbit antibody against eIF4A , polyclonal rabbit antibody against eIF4B , polyclonal rabbit antibody against rps16 ). Immunocomplexes were detected using a ECL kit  according to the manufacturer’s recommendation.Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked for 1 h at RT with 5% milk and incubated overnight in TBS-T  supplemented with BSA (5%) and with appropriate antibodies , polyclonal rabbit antibody against phospho-mTOR2, 2 mM DTT, 0.25% Nonidet P-40, 0.2% SDS and protease inhibitors cocktail. Cell debris was removed by centrifugation at 10,000 rpm for 15 min, and extracts were then directly utilized for IP. For IP, cell extracts were incubated with anti-YB-1 antibodies (approximately 100 μg) immobilized on protein G-Sepharose beads  for 2 h at 4 °C. After extensive washing with PBS, the proteins were eluted with acid-urea sample buffer , and analyzed by acid-urea 10% polyacrylamide gel electrophoresis (AUE) and autoradiography. Relative radioactivity of the bands or lanes was determined using a Packard Cyclone Storage Phosphor System . The level of total protein synthesis or YB-1 synthesis in untreated cells was taken to be 100%.Cells were washed with phosphate buffered saline, scraped and lysed with buffer containing 20 mM Hepes-KOH, pH 7.6, 100 KCl, 5 mM MgCl4 in 75 mM NaOAc buffer (pH 4.5). The reaction was performed in the dark at 4 °C for 45 min. After ethanol precipitation, the RNA was dissolved in 100 μl of fresh solution of 10 mM biotin hydrazide  in dimethyl sulfoxide. Then the samples were incubated for 4 h at 37 °C, and each of them was supplemented with 100 μl of 200 mM NaBH4 and 200 μl of 1 M Tris-HCl (pH 8.0) with subsequent incubation in the dark at 4 °C for 30 min. Then the RNA was ethanol-precipitated and dissolved in the required volume of H2O.3′-end biotinylation was performed as described previously7G Capping System and ScriptCap 2′-O-Methyltransferase Enzyme (CellScript) according to the suppliers’ recommendations.After 3′-biotinylation RNA fragments were capped using a ScriptCap™ mE. coli up to 200 μg/ml) at 4 °C with constant stirring. After incubation, the resin was washed five times with 10 volumes of PBS and 2 times with 10 volumes of buffer for in vitro translation 2, 8 mM creatine phosphate, 1 mM ATP, 0.2 mM GTP, 100 mM KAc, and 25 μM of each amino acid). The 2 pmol capped biotinylated RNA was incubated with 40 μl HeLa cell lysate in in vitro translation buffer supplemented with 20 μg of total E. coli RNA for 15 min at 30 °C. This mixture was then supplemented with 35 μl of 50% pre-blocked Streptavidin-Sepharose slurry and incubated for 1 h at room temperature. The resin with RNA–protein complexes was washed 8 times with 30 volumes of PBS (with up to 0.2 mM of VRC added). Bound proteins were eluted with the same buffer as for SDS-PAGE , separated by 10% SDS-PAGE and analyzed by Western blotting.Streptavidin-Sepharose  was washed five times with 30 volumes of PBS supplemented with up to 0.2 mM VRC . Then the resin was incubated overnight with a blocking solution .