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• W2030405536 abstract "In eubacteria, the dissociation of the 70 S ribosome into the 30 S and 50 S subunits is the essential first step for the translation initiation of canonical mRNAs that possess 5′-leader sequences. However, a number of leaderless mRNAs that start with the initiation codon have been identified in some eubacteria. These have been shown to be translated efficiently in vivo. Here we investigated the process by which leaderless mRNA translation is initiated by using a highly reconstituted cell-free translation system from Escherichia coli. We found that leaderless mRNAs bind preferentially to 70 S ribosomes and that the leaderless mRNA·70 S·fMet-tRNA complex can transit from the initiation to the elongation phase even in the absence of initiation factors (IFs). Moreover, leaderless mRNA translation proceeds more efficiently if the intact 70 S ribosome is involved compared with the 30 S subunit. Furthermore, excess amounts of IF3 inhibit leaderless mRNA translation, probably because it promotes the disassembly of the 70 S ribosome into subunits. Finally, excess amounts of fMet-tRNA facilitate the IF-independent translation of leaderless mRNA. These observations strongly suggest that leaderless mRNA translation is initiated by the assembled 70 S ribosome and thereby bypasses the dissociation process. In eubacteria, the dissociation of the 70 S ribosome into the 30 S and 50 S subunits is the essential first step for the translation initiation of canonical mRNAs that possess 5′-leader sequences. However, a number of leaderless mRNAs that start with the initiation codon have been identified in some eubacteria. These have been shown to be translated efficiently in vivo. Here we investigated the process by which leaderless mRNA translation is initiated by using a highly reconstituted cell-free translation system from Escherichia coli. We found that leaderless mRNAs bind preferentially to 70 S ribosomes and that the leaderless mRNA·70 S·fMet-tRNA complex can transit from the initiation to the elongation phase even in the absence of initiation factors (IFs). Moreover, leaderless mRNA translation proceeds more efficiently if the intact 70 S ribosome is involved compared with the 30 S subunit. Furthermore, excess amounts of IF3 inhibit leaderless mRNA translation, probably because it promotes the disassembly of the 70 S ribosome into subunits. Finally, excess amounts of fMet-tRNA facilitate the IF-independent translation of leaderless mRNA. These observations strongly suggest that leaderless mRNA translation is initiated by the assembled 70 S ribosome and thereby bypasses the dissociation process. Translation initiation plays a pivotal role in the translational control of gene expression. Although the details of the mechanism that initiates translation vary among eukaryotes, prokaryotes, and archae, the productivity of translation in general is largely dependent on the formation of the initiation complex, which involves the binding of the mRNA to the ribosome and the recognition of the initiation codon. In eubacteria, translation initiation involves three steps. First, the 70 S ribosome is induced to dissociate into the 30 S and 50 S subunits by initiation factor (IF) 1The abbreviations used are: IF, initiation factor; DHFR or dhfr, dihydrofolic acid reductase; EF, elongation factor; Eps, epsilon; fMet-tRNA, formylated methionyl-tRNAfMet; RF, release factor; SD, Shine-Dalgarno.1The abbreviations used are: IF, initiation factor; DHFR or dhfr, dihydrofolic acid reductase; EF, elongation factor; Eps, epsilon; fMet-tRNA, formylated methionyl-tRNAfMet; RF, release factor; SD, Shine-Dalgarno. 3 and IF1. Second, mRNA and the fMet-tRNA·IF2 complex bind the 30 S subunit in a random order (1Wu X.Q. Iyengar P. RajBhandary U.L. EMBO J. 1996; 15: 4734-4739Crossref PubMed Scopus (29) Google Scholar), which results in the formation of the 30 S initiation complex. Finally, the 50 S subunit joins the 30 S initiation complex, thereby forming the 70 S initiation complex that is now set for the transition into the elongation phase (for review, see Refs. 2Gualerzi C.O. Pon C.L. Biochemistry. 1990; 29: 5881-5889Crossref PubMed Scopus (400) Google Scholar and 3Gualerzi C.O. Brandi L. Caserta E. La Teana A. Spurio R. Tomsic J. Pon C.L. Grrett R.A. Douthwaite S.R. Liljas A. Matheson A.T. Moore P.B. Noller H.F. The Ribosome: Structure, Function, Antibiotics and Cellular Interactions. American Society for Microbiology, Washington, D. C.2000: 477-494Google Scholar). As indicated, three IFs, namely, IF1, IF2, and IF3, modulate the kinetics of these processes. IF2 conveys fMet-tRNA onto the ribosomal P site (2Gualerzi C.O. Pon C.L. Biochemistry. 1990; 29: 5881-5889Crossref PubMed Scopus (400) Google Scholar, 4Hartz D. McPheeters D.S. Gold L. Genes Dev. 1989; 3: 1899-1912Crossref PubMed Scopus (190) Google Scholar) and promotes the subsequent association of the 30 S and 50 S subunits (5La Teana A. Gualerzi C.O. Dahlberg A.E. RNA. 2001; 7: 1173-1179Crossref PubMed Scopus (67) Google Scholar). IF3 acts as a dissociation and fidelity factor that binds the 30 S subunit to prevent it from rejoining the 50 S subunit and ensures the proper positioning of the start codon and the initiator tRNA in the P site (4Hartz D. McPheeters D.S. Gold L. Genes Dev. 1989; 3: 1899-1912Crossref PubMed Scopus (190) Google Scholar, 6Dallas A. Noller H.F. Mol. Cell. 2001; 8: 855-864Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 7Petrelli D. LaTeana A. Garofalo C. Spurio R. Pon C.L. Gualerzi C.O. EMBO J. 2001; 20: 4560-4569Crossref PubMed Scopus (93) Google Scholar). IF1 promotes the activities of IF2 and IF3 (2Gualerzi C.O. Pon C.L. Biochemistry. 1990; 29: 5881-5889Crossref PubMed Scopus (400) Google Scholar) and protects the A site together with IF2 during the initiation event (8Brock S. Szkaradkiewicz K. Sprinzl M. Mol. Microbiol. 1998; 29: 409-417Crossref PubMed Scopus (64) Google Scholar, 9Carter A.P. Clemons Jr., W.M. Brodersen D.E. Morgan-Warren R.J. Hartsch T. Wimberly B.T. Ramakrishnan V. Science. 2001; 291: 498-501Crossref PubMed Scopus (305) Google Scholar).The processes described above have been studied intensively using canonical mRNAs that bear 5′-leader sequences containing a ribosome binding site such as the Shine-Dalgarno (SD) sequence (10Shine J. Dalgarno L. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 1342-1346Crossref PubMed Scopus (2552) Google Scholar). However, mRNAs that lack 5′-leader sequences do occur in substantial numbers in mainly eubacteria and archae (11Janssen G.R. Baltz R.H. Hegeman G.D. Skatrud P.L. Industrial Microorganisms: Basic and Applied Molecular Genetics. American Society for Microbiology, Washington, D. C.1993: 59-67Google Scholar, 12Moll I. Grill S. Gualerzi C.O. Blasi U. Mol. Microbiol. 2002; 43: 239-246Crossref PubMed Scopus (165) Google Scholar). Such mRNAs, which are termed leaderless mRNAs, are translated efficiently in vivo despite of the lack of obvious ribosome binding sites (11Janssen G.R. Baltz R.H. Hegeman G.D. Skatrud P.L. Industrial Microorganisms: Basic and Applied Molecular Genetics. American Society for Microbiology, Washington, D. C.1993: 59-67Google Scholar, 13Wu C.J. Janssen G.R. Mol. Microbiol. 1996; 22: 339-355Crossref PubMed Scopus (41) Google Scholar, 14Van Etten W.J. Janssen G.R. Mol. Microbiol. 1998; 27: 987-1001Crossref PubMed Scopus (63) Google Scholar). Previous studies examining the mechanism by which leaderless mRNA translation is initiated have shown that the binding of fMet-tRNA to the ribosome is a prerequisite for the binding of leaderless mRNAs to the ribosome (15Balakin A.G. Skripkin E.A. Shatsky I.N. Bogdanov A.A. Nucleic Acids Res. 1992; 20: 563-571Crossref PubMed Scopus (53) Google Scholar, 16Grill S. Gualerzi C.O. Londei P. Blasi U. EMBO J. 2000; 19: 4101-4110Crossref PubMed Scopus (104) Google Scholar). It was also demonstrated that IF2 selectively stimulates the translation of leaderless mRNA both in vitro and in vivo (16Grill S. Gualerzi C.O. Londei P. Blasi U. EMBO J. 2000; 19: 4101-4110Crossref PubMed Scopus (104) Google Scholar, 17Grill S. Moll I. Hasenohrl D. Gualerzi C.O. Blasi U. FEBS Lett. 2001; 495: 167-171Crossref PubMed Scopus (41) Google Scholar), whereas IF3 discriminates against the 5′-terminal start codon of leaderless mRNA in the formation of the 30 S initiation complex (18Moll I. Resch A. Blasi U. FEBS Lett. 1998; 436: 213-217Crossref PubMed Scopus (35) Google Scholar, 19Tedin K. Moll I. Grill S. Resch A. Graschopf A. Gualerzi C.O. Blasi U. Mol. Microbiol. 1999; 31: 67-77Crossref PubMed Scopus (69) Google Scholar) and inhibits its translation in vivo (19Tedin K. Moll I. Grill S. Resch A. Graschopf A. Gualerzi C.O. Blasi U. Mol. Microbiol. 1999; 31: 67-77Crossref PubMed Scopus (69) Google Scholar). Furthermore, the IF3-dependent discrimination against the 5′-terminal start codon requires the ribosomal protein S1 (18Moll I. Resch A. Blasi U. FEBS Lett. 1998; 436: 213-217Crossref PubMed Scopus (35) Google Scholar). These studies together suggest that the formation of a 30 S initiation complex bearing leaderless mRNA may involve a 30 S subunit that lacks IF3 and/or S1. However, contradictory studies show that leaderless mRNAs prefer to bind to intact 70 S ribosomes rather than to the 30 S subunit (15Balakin A.G. Skripkin E.A. Shatsky I.N. Bogdanov A.A. Nucleic Acids Res. 1992; 20: 563-571Crossref PubMed Scopus (53) Google Scholar, 20O'Donnell S.M. Janssen G.R. J. Bacteriol. 2002; 184: 6730-6733Crossref PubMed Scopus (56) Google Scholar, 21Tedin K. Resch A. Blasi U. Mol. Microbiol. 1997; 25: 189-199Crossref PubMed Scopus (59) Google Scholar), which suggests that the translation initiation of leaderless mRNA may actually involve an intact 70 S ribosome. This would give leaderless mRNAs a competitive advantage in vivo because the dissociation of the 70 S ribosome into the 30 S and 50 S subunits appears to be the essential step for the initiation of canonical mRNA translation. These studies indicate that the mechanism that initiates the translation of leaderless mRNA is still unclear.Recently, Shimizu et al. (22Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Nat. Biotechnol. 2001; 19: 751-755Crossref PubMed Scopus (1289) Google Scholar) constructed a cell-free translation system that is reconstituted of purified components and is denoted the PURE system. The PURE system is composed of only those factors that are essential for the translation of natural mRNAs. This system thus allows us to adjust the concentration of each component and to pause the translation reaction at a particular step by omitting a component that is essential for that step. These advantages enable us to evaluate the involvement of a particular factor in translation more clearly than is possible with the other cell-free translation systems, such as the Escherichia coli S30 extract system. By using this PURE system, we here provide experimental evidence showing that translation of leaderless mRNA is initiated by direct binding by the mRNA to the intact 70 S ribosome and that subunit dissociation followed by mRNA association with the 30 S subunit, as is required for the translation of canonical mRNAs in eubacteria, is not involved.EXPERIMENTAL PROCEDURESPreparation of mRNAs—The mRNAs used in this study were synthesized by in vitro transcription using T7 RNA polymerase. The templates for transcription were prepared as follows. The constructs encoding dihydrofolic acid reductase (DHFR) are shown in Table I. These include dhfr-Eps, dhfr-SD, dhfr-AUG, dhfr-plus5, and dhfr-plus10. The template for dhfr-Eps (pUC-dhfr-Eps) is identical to a model template used in a previous study (22Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Nat. Biotechnol. 2001; 19: 751-755Crossref PubMed Scopus (1289) Google Scholar). The template for dhfr-SD (pUC-dhfr-SD), which contains a 5′-leader sequence that is identical to that of wild-type DHFR mRNA, was amplified from pUC-dhfr-Eps by PCR with the following primers: T7dhfr5′-SD-BamHI (5′-CGCGGATCCGTAATACGACTCACTATAGGGAATTTTTTTTATCGGGAAATCT-3′) and dhfr3′-PstI (5′-AAAACTGCAGCTTACCGCCGCTCCAGAAT-3′). The amplification product was then cloned into pUC18. The construct for dhfr-AUG (pUC-dhfr-AUG), which has only a single nucleotide upstream of the start codon, was prepared in the same way as dhfr-SD except that the following primers were used: T7dhfr5′-AUG-BamHI (5′-CGCGGATCCGTAATACGACTCACTATAGATGATCAGTCTGATTGCGG-3′) and dhfr3′-PstI. These amplification products were also cloned into pUC18. The constructs for dhfr-plus5 and dhfr-plus10 were subsequently generated from pUC-dhfr-AUG by the QuikChange site-directed mutagenesis kit (Stratagene) with the following primer pairs: for dhfr-plus5, 5′-CTATAGCTCAATGATCAGTCTGATTGCGGC-3′ (forward) and 5′-TGAGCTATAGTGAGTCGTATTACGGATCCC-3′ (backward); and for dhfr-plus10, 5′-GTCCCTCTCAATGATCAGTCTGATTGCGGC-3′ (forward) and 5′-TGAGAGGGACTATAGTGAGTCGTATTACGG-3′ (backward). The gene for the λcI repressor was amplified from λDNA by PCR with the following primers: cI5′EcoRI (5′-AATAAGAATTCTAATACGACTCACTATAGATGAGCACAAAAAAGAAACCA-3′) and cI3′uaaSma-IPstI (5′-AATAACTGCAGCCCGGGTTAGCCAAACGTCTCTTCAG-3′). The amplification products were cloned into pUC18, resulting in pUC-cI. All constructs contained T7 promoter sequence upstream of their 5′-leader sequences. After digestion by SmaI or PstI, the linearized templates were used for in vitro transcription. The transcription products were purified by Qiagen-tip 20 (Qiagen) according to the manufacturer’s instructions.Table INucleotide sequences around the start codon of the mRNAs used in this studymRNANucleotide sequences around the start codonaSD sequences are underlined. Start codons are marked in bold.dhfr-Eps5′-GGGUCUAGAGUUUAACUUUAAGAAGGAGA UAUACAUAUGAUCAGUCUGAUU-3′dhfr-SD5′-GGGAAUUUUUUUUAUCGGGAAAUCUCAAUGAUCAGUCUGAUU-3′dhfr-AUG5′-GAUGAUCGAUCUGAUU-3′dhfr-plus55′-GCUCAAUGAUCAGUCUGAUU-3′dhfr-plus105′-GUCCCTCUCAAUGAUCAGUCUGAUU-3′cI5′-GAUGAGCACAAAAAAG-3′a SD sequences are underlined. Start codons are marked in bold. Open table in a new tab Preparation of Ribosomes—Tight coupled 70 S ribosomes were purified from E. coli MRE600 cells by 6–38% sucrose density gradient centrifugation according to the method of Spedding (23Spedding G. Spedding G. Ribosome and Protein Synthesis: A Practical Approach. IRL Press, New York1990: 1-29Google Scholar). Prior to sucrose density gradient, crude ribosomes were salt-washed twice with 0.5 m NH4Cl. It was confirmed by SDS-PAGE and Western blotting that the prepared 70 S ribosome was essentially free of IFs. The 30 S and 50 S subunits were subsequently prepared from the 70 S ribosome by sucrose density gradient with low magnesium concentration (1 mm) buffers. The S1-free 70 S ribosome was prepared by a poly(U)-Sepharose column (Amersham Biosciences) as described previously (24Suryanarayana T. Subramanian A.R. Biochemistry. 1983; 22: 2715-2719Crossref PubMed Scopus (30) Google Scholar).Preparation of Translation Factors—His6-tagged translation factors, apart from the IFs, were purified as described previously (22Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Nat. Biotechnol. 2001; 19: 751-755Crossref PubMed Scopus (1289) Google Scholar). The genes encoding IF1, IF2, and IF3 were amplified by PCR from the genome of E. coli A19 using the following primer pairs: for IF1, 5′-GGGAATTCCATATGGCCAAAGAAGACAATATTG-3′ and 5′-CGCGGATCCTTAGCGACTACGGAAGACAATG-3′; for IF2, 5′-GGGAATTCCATATGACAGATGTAACGATTAAAAC-3′ and 5′-CGCGGATCCTTAAGCAATGGTACGTTGGATC-3′; and for IF3, 5′-GGGAATTCCATATGAAAGGCGGAAAACGAGTTC-3′ and 5′-CGCGGATCCTTACTGTTTCTTCTTAGGAGCG-3′. The PCR products were cloned into pET17b (Novagen), which produces recombinant factors without His6 tags. IF2 and IF3 were overexpressed in E. coli BL21(DE3) cells and purified as described (25Soffientini A. Lorenzetti R. Gastaldo L. Parlett J.H. Spurio R. La Teana A. Islam K. Protein Expr. Purif. 1994; 5: 118-124Crossref PubMed Scopus (35) Google Scholar). IF1 was purified by the same procedure used for IF3 followed by gel filtration chromatography using Sephadex 75 HR10/30 (Amersham Biosciences) with HT buffer containing 50 mm HEPESKOH, pH 7.6, 200 mm KCl, 10 mm MgCl2, 7 mm β-mercaptoethanol, and 10% glycerol. The purified factors were dialyzed against HT buffer containing 30% glycerol and stored at –80 °C.In Vitro Translation (PURE System)—The system was reconstituted as published previously (22Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Nat. Biotechnol. 2001; 19: 751-755Crossref PubMed Scopus (1289) Google Scholar) with slight modifications. Briefly, in standard conditions, the system was composed of 0.5 μm 70 S ribosomes, 0.5 μm each IF1, IF2, and IF3, 2 μm EF-Tu, 1 μm EF-Ts, 0.5 μm EF-G, 0.25 μm each RF1 and RF3, 0.5 μm ribosome recycling factor, 30–300 units of each aminoacyl-tRNA synthetase, methionyl-tRNA formyltransferase, 3 μg/ml myokinase (Sigma), 1.08 μg/ml nucleoside diphosphate kinase, 30 units/ml inorganic pyrophosphatase (Sigma), 4 μg/ml creatine kinase (Roche Applied Science), 20 mm creatine phosphate, 50 μm tRNA mixture (Roche Applied Science), 0.1 mm concentration of each amino acid, and 4 MBq/ml [35S]methionine (Muromachi). The other components were the same as described previously (22Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Nat. Biotechnol. 2001; 19: 751-755Crossref PubMed Scopus (1289) Google Scholar). The reactions were initiated by adding 0.5 μm mRNA. After incubation at 37 °C for 1 h, 5 μl of the mixture was withdrawn and mixed with an equal volume of SDS-PAGE sample loading buffer. The proteins that were synthesized were separated by 12% SDS-PAGE. The gels were then dried and exposed to an imaging plate followed by analysis by a BAS5000 bioimaging analyzer (Fujifilm). The assay conditions for single turnover reactions (see Figs. 3 and 4) are described in each figure legend.Fig. 4Dependence of leaderless and leadered mRNA translation on IF3.A, in vitro translation of cI (open squares) and dhfr-Eps (open circles) mRNA. The reactions were performed as described under “Experimental Procedures,” although the concentration of IF3 varied. Translation of cI mRNA using an S1-free 70 S ribosome is shown by the closed squares. The products were analyzed by 12% SDS-PAGE. The protein synthesis of cI and DHFR at the IF3 concentration of 0.5 μm was set to 1. B, translation of dhfr-SD (open circles) and dhfr-AUG (open squares) mRNA. The reactions were performed as described in A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Filter Binding Assay—Filter binding assays were performed according to Tedin et al. (21Tedin K. Resch A. Blasi U. Mol. Microbiol. 1997; 25: 189-199Crossref PubMed Scopus (59) Google Scholar) with slight modifications. mRNAs were labeled with [γ-32P]ATP by T4 polynucleotide kinase. To allow the 30 S and 50 S subunits to associate, 10 pmol of the 30 S subunit was preincubated at 37 °C for 5 min with an equal molar amount of the 50 S subunit in 50 μl of PM buffer containing 5 mm potassium phosphate, pH 7.3, 95 mm KCl, 5 mm NH4Cl, 0.5 mm CaCl2, 6 mm MgCl2, 1 mm spermidine, 8 mm putrescine, and 1 mm dithiothreitol. Thereafter, 1 pmol of 32P-labeled mRNA was added to the mixture, after which fMet-tRNA and the IFs were added in a 5-fold excess and at an equal molar amount relative to the ribosome amount, respectively. It was confirmed that the concentrations mentioned above did not induce higher than background levels of nonspecific binding of mRNA to the IFs. After an incubation for 10 min at 37 °C, the samples were filtered through nitrocellulose membrane and washed three times with 1.5 ml of cold PM buffer. The filters were then dried and exposed to an imaging plate. The percentages of retained mRNAs were normalized by setting the radioactivity of the total input mRNA at 100%.RESULTSTranslation of Leaderless mRNAs by the PURE System—The PURE system is suitable for the analysis of individual steps of the translation process because it is a minimal system composed of only the essential translation factors (22Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Nat. Biotechnol. 2001; 19: 751-755Crossref PubMed Scopus (1289) Google Scholar). Initially, we confirmed by using the PURE system that leaderless mRNAs can be translated, as has been revealed previously by in vivo experiments (14Van Etten W.J. Janssen G.R. Mol. Microbiol. 1998; 27: 987-1001Crossref PubMed Scopus (63) Google Scholar, 16Grill S. Gualerzi C.O. Londei P. Blasi U. EMBO J. 2000; 19: 4101-4110Crossref PubMed Scopus (104) Google Scholar, 26Jones III, R.L. Jaskula J.C. Janssen G.R. J. Bacteriol. 1992; 174: 4753-4760Crossref PubMed Google Scholar). The mRNAs used in this study are described in Table I. The mRNA encoding dhfr-Eps is the model mRNA for the PURE system and was highly translated in the system by virtue of both strong SD and Eps sequences in its 5′-leader region (Fig. 1, lane 1). As shown in Fig. 1, both the naturally occurring leaderless λ phage cI mRNA (lane 6) and the unnatural leaderless dhfr-AUG mRNA (lane 3) were also translated efficiently. The translation efficiency of dhfr-AUG mRNA was about half of that of the dhfr-SD mRNA (lane 2), whose upstream sequence is identical to that of the DHFR gene in the E. coli genome. However, the addition of 5–10 bases to the 5′-end of dhfr-AUG seriously hindered the translation reaction (lanes 4 and 5). It is likely that the additional bases in dhfr-plus5 and dhfr-plus10 mRNA abrogate the correct positioning of the start codon in the P site. These results are almost equivalent to the observations made with in vivo experiments (14Van Etten W.J. Janssen G.R. Mol. Microbiol. 1998; 27: 987-1001Crossref PubMed Scopus (63) Google Scholar, 26Jones III, R.L. Jaskula J.C. Janssen G.R. J. Bacteriol. 1992; 174: 4753-4760Crossref PubMed Google Scholar). Moreover, it suggests that leaderless mRNA translation does not require factors other than those in the PURE system, at least not leaderless mRNA translation in E. coli.Fig. 1Translation of leadered and leaderless mRNAs by the PURE system. The mRNAs shown in Table I were translated by the PURE system and analyzed on a 12% SDS-polyacrylamide gel as described under “Experimental Procedures.” Lanes 1–6 indicate the translation products of the dhfr-Eps, dhfr-SD, dhfr-AUG, dhfr-plus5, dhfr-plus10, and cI mRNAs, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Leaderless and Leadered mRNA Binding to the 30 S Subunit or the 70 S Ribosome—Some natural leaderless mRNAs, such as those encoding λ phage cI, P2 phage gene V, or Tn1721 TetR, have been shown to bind preferentially to the 70 S ribosome compared with the 30 S subunit (15Balakin A.G. Skripkin E.A. Shatsky I.N. Bogdanov A.A. Nucleic Acids Res. 1992; 20: 563-571Crossref PubMed Scopus (53) Google Scholar, 20O'Donnell S.M. Janssen G.R. J. Bacteriol. 2002; 184: 6730-6733Crossref PubMed Scopus (56) Google Scholar, 21Tedin K. Resch A. Blasi U. Mol. Microbiol. 1997; 25: 189-199Crossref PubMed Scopus (59) Google Scholar). The selective preference of leaderless mRNA for the 70 S ribosome or the 30 S subunit can be evaluated by the filter binding assay, which employs purified ribosomes from which adherent IFs have been removed (Fig. 2). The reactions all contained an excess of fMet-tRNA. The contribution IFs make to mRNA-ribosome binding was assessed by conducting the experiment in the presence or absence of IFs. As was expected, in the absence of IFs, the leaderless cI mRNA bound significantly better to the 70 S ribosome (16%) than to the 30 S subunit (3.1%). The addition of the IFs enhanced the leaderless mRNA binding to the 30 S subunit up to 12%, but even in these conditions, the cI mRNA still showed a slight preference for binding to the 70 S ribosome (19%). In the case of the unnatural leaderless mRNA dhfr-AUG, its ability to bind to the 70 S and the 30 S ribosomes was generally weaker or less stable than that of the cI mRNA. Nevertheless, it had the same binding features as cI mRNA because dhfr-AUG mRNA formed the mRNA·70 S·fMet-tRNA initiation-like complex in the presence or absence of IFs. Therefore, it is possible to say that in the presence of fMet-tRNA, leaderless mRNAs prefer to bind to the 70 S ribosome than to the 30 S subunit. However in the absence of fMet-tRNA, leaderless mRNAs were able to bind neither to 30 S nor to 70 S ribosome, probably because these cannot be tethered by ribosome in the absence of base pairing between 5′-terminal start codon and anti-codon of fMet-tRNA. It is plausible that in the presence of IFs, leadered mRNA binds to 70 S ribosome as well as to 30 S subunit because IF3 induce subunit dissociation. Unexpectedly, the leadered dhfr-Eps and dhfr-SD mRNAs bound not only the 30 S subunit, they also associated with the 70 S ribosome even in the absence of IFs (Fig. 2). However, as will be shown below, these leadered mRNA·70 S·fMet-tRNA complexes, unlike the leaderless mRNA·70 S·fMet-tRNA complexes, are not able to support translation in the absence of IFs.Fig. 2The ability of dhfr-Eps, dhfr-SD, dhfr-AUG, and cI mRNA to bind to the 30 S subunit or the 70 S ribosome. The radiolabeled mRNA levels retained with the ribosome were measured by the filter binding assay (see “Experimental Procedures”). The assay conditions used are described below each bar. The molar ratio of the ribosomes, the mRNA, the fMet-tRNA, and the IFs was 10:1:50:10 when both fMet-tRNA and IFs were included.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Translation Efficiency of Leaderless and Leadered mRNA When Associated with the 30 S Subunit or the 70 S Ribosome—We next examined whether the 70 S initiation-like complexes that are formed with the leaderless mRNA in the presence or absence of IFs are functionally active with regard to translation and whether the translation initiation of leaderless mRNA proceeds via the 30 S subunit or via the undissociated 70 S ribosome. The experimental scheme employed is depicted in Fig. 3A. The mRNA species was first preincubated with the 30 S subunit or the 70 S ribosome, together with fMet-tRNA, in the presence or absence of IFs. The resulting mixture contains the ternary complex that is composed of mRNA, ribosome, and fMet-tRNA. At the same time, the components for elongation (elongation factors, tRNAs, amino acids, [35S]methionine, aminoacyl-tRNA synthetases) were incubated to generate the aminoacyl-tRNAs. The peptide elongation reaction was then initiated by adding the elongation mixtures, and the 50 S subunit when initiation mixture does not include 50 S, to the initiation mixtures. These experiments reflect single turnover reactions because release factors, methionyl-tRNA formyltransferase, and formyl donors were omitted from the system, so the ribosome would stall at the stop codon. This experimental procedure allows us to clarify whether the mRNA·70 S·fMet-tRNA complex is capable of progressing into the elongation phase and whether the 30 S or the 70 S pathway is responsible for the translation of leadered and leaderless mRNAs.As shown in Fig. 3B, in the presence of IFs, cI mRNA was translated more efficiently when it was complexed to the 70 S ribosome than when it was complexed with the 30 S subunit. This indicates that the dissociation of the 70 S ribosome into subunits hinders the translation of cI mRNA. Moreover, when IFs were absent, cI mRNA was still translated to a considerable degree when it was complexed with the 70 S ribosome, whereas cI mRNA complexed with the 30 S subunit was not translated at all. Fig. 3D indicates that the translation of dhfr-AUG mRNA was also initiated more efficiently by the 70 S ribosome than by the 30 S subunit. In contrast, in the case of dhfr-Eps and dhfr-SD mRNA, the translation efficiencies initiated by the 30 S subunit and the 70 S ribosomes were almost the same in the presence of IFs (Fig. 3, C and E). Moreover, when IFs were absent, dhfr-Eps mRNA was considerably translated when complexed to the 30 S subunit, but its translation was poor when it was complexed to the 70 S ribosome (Fig. 3C). However, dhfr-SD mRNA was barely translated when it was initiated by either the 30 S subunit or the 70 S ribosome in the absence of IFs. It may be that in the case of dhfr-SD mRNA, the weak SD sequence in its upstream region may make the mRNA-30 S-fMet-tRNA bond too unstable to support the formation of a secure 30 S ternary complex (see Table I). Thus, IFs are indispensable for stabilizing the binding of dhfr-SD mRNA to the ribosome (see Fig. 2).Together with the results obtained in Fig. 2, these observations indicate that the leaderless mRNA·70 S·fMet-tRNA initiation-like complex is capable of progressing into the elongation phase. They also strongly suggest that the translation of leaderless mRNAs preferentially proceeds by the binding of the mRNA to the intact 70 S ribosome, thereby bypassing the dissociation of the subunits. In contrast, leadered mRNAs are translated by binding to the 30 S subunit in accordance with the canonical initiation process (2Gualerzi C.O. Pon C.L. Biochemistry. 1990; 29: 5881-5889Crossref PubMed Scopus (400) Google Scholar).Effect of IF" @default.
• W2030405536 created "2016-06-24" @default.
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• W2030405536 date "2004-03-01" @default.
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