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• W2078884107 abstract "When bound to Escherichia coliribosomes and irradiated with near-UV light, various derivatives of yeast tRNAPhe containing 2-azidoadenosine at the 3′ terminus form cross-links to 23 S rRNA and 50 S subunit proteins in a site-dependent manner. A and P site-bound tRNAs, whose 3′ termini reside in the peptidyl transferase center, label primarily nucleotides U2506 and U2585 and protein L27. In contrast, E site-bound tRNA labels nucleotide C2422 and protein L33. The cross-linking patterns confirm the topographical separation of the peptidyl transferase center from the E site domain. The relative amounts of label incorporated into the universally conserved residues U2506 and U2585 depend on the occupancy of the A and P sites by different tRNA ligands and indicates that these nucleotides play a pivotal role in peptide transfer. In particular, the 3′-adenosine of the peptidyl-tRNA analogue, AcPhe-tRNAPhe, remains in close contact with U2506 regardless of whether its anticodon is located in the A site or P site. Our findings, therefore, modify and extend the hybrid state model of tRNA-ribosome interaction. We show that the 3′-end of the deacylated tRNA that is formed after transpeptidation does not immediately progress to the E site but remains temporarily in the peptidyl transferase center. In addition, we demonstrate that the E site, defined by the labeling of nucleotide C2422 and protein L33, represents an intermediate state of binding that precedes the entry of deacylated tRNA into the F (final) site from which it dissociates into the cytoplasm. When bound to Escherichia coliribosomes and irradiated with near-UV light, various derivatives of yeast tRNAPhe containing 2-azidoadenosine at the 3′ terminus form cross-links to 23 S rRNA and 50 S subunit proteins in a site-dependent manner. A and P site-bound tRNAs, whose 3′ termini reside in the peptidyl transferase center, label primarily nucleotides U2506 and U2585 and protein L27. In contrast, E site-bound tRNA labels nucleotide C2422 and protein L33. The cross-linking patterns confirm the topographical separation of the peptidyl transferase center from the E site domain. The relative amounts of label incorporated into the universally conserved residues U2506 and U2585 depend on the occupancy of the A and P sites by different tRNA ligands and indicates that these nucleotides play a pivotal role in peptide transfer. In particular, the 3′-adenosine of the peptidyl-tRNA analogue, AcPhe-tRNAPhe, remains in close contact with U2506 regardless of whether its anticodon is located in the A site or P site. Our findings, therefore, modify and extend the hybrid state model of tRNA-ribosome interaction. We show that the 3′-end of the deacylated tRNA that is formed after transpeptidation does not immediately progress to the E site but remains temporarily in the peptidyl transferase center. In addition, we demonstrate that the E site, defined by the labeling of nucleotide C2422 and protein L33, represents an intermediate state of binding that precedes the entry of deacylated tRNA into the F (final) site from which it dissociates into the cytoplasm. N-acetylphenylalanyl-tRNAPhe polyacrylamide gel electrophoresis elongation factor Tu guanylyl-imidodiphosphate Knowledge of the molecular events that take place during protein synthesis has been greatly influenced by studies demonstrating that aminoacyl-tRNA, peptidyl-tRNA, and deacylated tRNA are accommodated on the ribosome at the A, P, and E sites, respectively (reviewed in Ref.1Wower J. Zimmermann R.A. Biochimie (Paris). 1991; 73: 961-969Crossref PubMed Scopus (18) Google Scholar). The three-site model of translation has been modified to include three hybrid binding states, designated A/T, A/P, and P/E, which are adopted by tRNA during its passage through the ribosome (2Moazed D. Noller H.F. Nature. 1989; 342: 142-148Crossref PubMed Scopus (608) Google Scholar). Our earlier photoaffinity labeling studies, which focused primarily on the identification of ribosomal proteins contacted by the 3′ end and anticodon of tRNA as it transits the ribosome, allowed us to propose a model for the arrangement of the A, P, and E-site tRNAs on the Escherichia coli ribosome (3Wower J. Hixson S.S. Zimmermann R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5232-5236Crossref PubMed Scopus (96) Google Scholar, 4Wower J. Scheffer P. Sylvers L.A. Wintermeyer W. Zimmermann R.A. EMBO J. 1993; 12: 617-623Crossref PubMed Scopus (39) Google Scholar). In this model, the tRNA molecules are positioned so that their 3′ ends are directed toward the peptidyl transferase center of the 50 S ribosomal subunit, while their anticodons point toward the groove between the head and the body of the 30 S ribosomal subunit where they interact with complementary codons in the mRNA. Relative to the 50 S subunit interface, the A-site tRNA is located on the L7/L12 or “right” side, the E site tRNA is placed near the L1 protuberance on the “left” side, and the P- site tRNA occupies the space between them. A similar model was proposed by Noller et al. (5Noller H.F. Moazed D. Stern S. Powers T. Allen P.N. Robertson J.M. Weiser B. Triman K. Hill W.E. Dahlberg A. Garrett R.A. Moore P.B. Schlessinger D. Warner J.R. The Ribosome: Structure, Function and Evolution. ASM Press, Washington, DC1990: 73-92Google Scholar), who investigated the location of tRNA in ribosomal complexes by chemical footprinting. Recently, cryo-electron microscopy and x-ray crystallography have permitted the visualization of tRNA molecules bound to the ribosome during different stages of protein synthesis at high resolution (6Agrawal R.K. Penczek P. Grassucci R.A. Li Y. Leith A. Nierhaus K.H. Frank J. Science. 1996; 271: 1000-1002Crossref PubMed Scopus (191) Google Scholar, 7Stark H. Orlova E.V. Rinke-Appel J. Junke N. Mueller F. Rodnina M. Wintermeyer W. Brimacombe R. van Heel M. Cell. 1997; 88: 19-28Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 8Cate J.H. Yusupov M.M. Yusupova G.Zh. Earnest T.N. Noller H.F. Science. 1999; 285: 2095-2104Crossref PubMed Scopus (524) Google Scholar). Stark et al. (7Stark H. Orlova E.V. Rinke-Appel J. Junke N. Mueller F. Rodnina M. Wintermeyer W. Brimacombe R. van Heel M. Cell. 1997; 88: 19-28Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) have demonstrated that the arrangement of the A, P, and E site tRNAs in pre- and postranslocational ribosomes is very close to that predicted by Wower et al. (3Wower J. Hixson S.S. Zimmermann R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5232-5236Crossref PubMed Scopus (96) Google Scholar, 4Wower J. Scheffer P. Sylvers L.A. Wintermeyer W. Zimmermann R.A. EMBO J. 1993; 12: 617-623Crossref PubMed Scopus (39) Google Scholar) and Noller et al. (5Noller H.F. Moazed D. Stern S. Powers T. Allen P.N. Robertson J.M. Weiser B. Triman K. Hill W.E. Dahlberg A. Garrett R.A. Moore P.B. Schlessinger D. Warner J.R. The Ribosome: Structure, Function and Evolution. ASM Press, Washington, DC1990: 73-92Google Scholar). An alternative arrangement for E site-bound tRNA, designated as the E2 site, has been proposed on the basis of cryo-electron microscopy (6Agrawal R.K. Penczek P. Grassucci R.A. Li Y. Leith A. Nierhaus K.H. Frank J. Science. 1996; 271: 1000-1002Crossref PubMed Scopus (191) Google Scholar, 9Frank J. J. Struct. Biol. 1998; 124: 142-150Crossref PubMed Scopus (19) Google Scholar). This tRNA binding state most likely corresponds to the E site tRNA visualized by x-ray crystallography (8Cate J.H. Yusupov M.M. Yusupova G.Zh. Earnest T.N. Noller H.F. Science. 1999; 285: 2095-2104Crossref PubMed Scopus (524) Google Scholar). Many lines of evidence indicate that the 23 S rRNA is involved in essential ribosomal functions (10Noller H.F. Annu. Rev. Biochem. 1991; 60: 122-191Crossref Scopus (413) Google Scholar, 11Muth G.W. Ortoleva-Donnelly L. Strobel S.A. Science. 2000; 289: 947-950Crossref PubMed Scopus (238) Google Scholar). Now that the position of tRNA molecules can be visualized relative to the structure of the ribosome, determining the topography of tRNA-23 S rRNA contacts on the level of individual nucleotides is crucial for interpreting the images provided by cryo-electron microscopy and x-ray crystallography as well as for constructing high resolution models of the different functional states of the ribosome. In the present work, we follow the movement of the 3′ terminus of tRNA as it transits the E. coli ribosome during the elongation cycle of translation using photoreactive tRNA probes and characterize the interactions of tRNA with the 23 S rRNA at the nucleotide level. The sources of tRNAs, enzymes, and radioactively labeled compounds were as described previously (4Wower J. Scheffer P. Sylvers L.A. Wintermeyer W. Zimmermann R.A. EMBO J. 1993; 12: 617-623Crossref PubMed Scopus (39) Google Scholar, 12Boon K. Vijgenboom E. Madsen L.V. Talens A. Kraal B. Bosch L. Eur. J. Biochem. 1992; 210: 177-183Crossref PubMed Scopus (54) Google Scholar, 13Wower J. Hixson S.S. Zimmermann R.A. Biochemistry. 1988; 27: 8114-8121Crossref PubMed Scopus (32) Google Scholar). [5′-32P](2N3A76)tRNAPheand [14C]tRNAPhe were prepared according to earlier described procedures (14Sylvers L. Wower J. Hixson S.S. Zimmermann R.A. FEBS Lett. 1989; 245: 9-13Crossref PubMed Scopus (21) Google Scholar, 15Semenkov Yu. P. Makarov E.M. Kirillov S.V. Biopolym. Cell. 1985; 1: 183-193Crossref Google Scholar). Aminoacylation of [5′-32P](2N3A76)tRNAPhe with [14C]Phe or [3H]Phe and acetylation of the aminoacylated photoreactive tRNA derivative were carried out according to Rappaport and Lapidot (16Rappaport S. Lapidot Y. Methods Enzymol. 1974; 29E: 685-693Crossref Scopus (61) Google Scholar). All Phe- and AcPhe-tRNAPhe1 derivatives were purified by benzoylated DEAE-cellulose chromatography (15Semenkov Yu. P. Makarov E.M. Kirillov S.V. Biopolym. Cell. 1985; 1: 183-193Crossref Google Scholar). Tight-couple 70 S ribosomes, isolated from E. coli MRE 600 as described by Makhno et al. (17Makhno V.I. Peshin N.N. Semenkov Yu. P. Kirillov S.V. Mol. Biol. 1988; 22: 528-536Google Scholar), bound 1.8 molecules of AcPhe-tRNAPhe per ribosome at 15 mmMg2+. Poly(U) templates used in cross-linking experiments were prepared according to Kirillov et al. (18Kirillov S.V. Makhno V.I. Semenkov Y.P. Eur. J. Biochem. 1978; 89: 297-304Crossref PubMed Scopus (25) Google Scholar). Binding of [5′-32P](2N3A76)tRNAPhederivatives to the ribosomal P, A, R, and E sites was carried out according to established procedures (2Moazed D. Noller H.F. Nature. 1989; 342: 142-148Crossref PubMed Scopus (608) Google Scholar, 3Wower J. Hixson S.S. Zimmermann R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5232-5236Crossref PubMed Scopus (96) Google Scholar, 17Makhno V.I. Peshin N.N. Semenkov Yu. P. Kirillov S.V. Mol. Biol. 1988; 22: 528-536Google Scholar, 19Semenkov Yu. P. Shapkina T.G. Kirillov S.V. FEBS Lett. 1992; 296: 207-210Crossref PubMed Scopus (40) Google Scholar) and measured by filter retention (20Nirenberg M. Leder P. Science. 1964; 145: 1399-1407Crossref PubMed Scopus (652) Google Scholar). Occupation of the P site by Ac[14C]Phe-[5′-32P](2N3A76)tRNAPhewas verified by its reactivity with puromycin (21Robertson J.M. Wintermeyer W. J. Mol. Biol. 1981; 151: 57-79Crossref PubMed Scopus (62) Google Scholar). Except as noted, tRNA binding and cross-linking experiments were carried out in TNME buffer (20 mm Tris-HCl, pH 7.5, 50 mmNH4Cl, 10 mm MgCl2, and 0.5 mm EDTA). Cross-linking was accomplished by irradiating non-covalent tRNA-ribosome complexes for 2 min at 0 °C in a Rayonet Model RPR-100 photochemical reactor equipped with six RPR-3000-Å lamps (13Wower J. Hixson S.S. Zimmermann R.A. Biochemistry. 1988; 27: 8114-8121Crossref PubMed Scopus (32) Google Scholar). On average, 9–13% of the non-covalently bound (2N3A76)tRNAPhe became covalently cross-linked to the ribosomes under our conditions (3Wower J. Hixson S.S. Zimmermann R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5232-5236Crossref PubMed Scopus (96) Google Scholar, 4Wower J. Scheffer P. Sylvers L.A. Wintermeyer W. Zimmermann R.A. EMBO J. 1993; 12: 617-623Crossref PubMed Scopus (39) Google Scholar, 13Wower J. Hixson S.S. Zimmermann R.A. Biochemistry. 1988; 27: 8114-8121Crossref PubMed Scopus (32) Google Scholar). The cross-linked complexes were fractionated as described previously (13Wower J. Hixson S.S. Zimmermann R.A. Biochemistry. 1988; 27: 8114-8121Crossref PubMed Scopus (32) Google Scholar, 22Dontsova O. Tishkov V. Dokudovskaya S. Bogdanov A. Döring T. Rinke-Appel J. Thamm S. Greuer B. Brimacombe R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4125-4129Crossref PubMed Scopus (64) Google Scholar). Sites in the 23 S rRNA labeled by (2N3A76)tRNAPhe were identified by treatment with RNase H in the presence of selected pairs of oligodeoxyribonucleotides (23Brimacombe R. Greuer B. Gulle H. Kosack M. Mitchell P. Osswald M. Stade K. Stiege W. Spedding G. Ribosomes and Protein Synthesis. A Practical Approach. Oxford University Press, Oxford1990: 131-159Google Scholar), RNase T1 protection experiments (24Rosen K.V. Alexander R.W. Wower J. Zimmermann R.A. Biochemistry. 1993; 32: 12802-12811Crossref PubMed Scopus (18) Google Scholar), and primer extension analysis (25Döring T. Mitchell P. Osswald M. Bochkariov D. Brimacombe R. EMBO J. 1994; 13: 2677-2685Crossref PubMed Scopus (88) Google Scholar, 26Moazed D. Stern S. Noller H.F. J. Mol. Biol. 1986; 187: 399-416Crossref PubMed Scopus (447) Google Scholar). The oligodeoxyribonucleotides used in these assays were complementary to sequences within the 23 S rRNA encompassing nucleotides 2776–2790 (oligo 1), 2616–2630 (oligo 2), 2582–2597 (oligo 2a), 2563–2577 (oligo 3), 2532–2546 (oligo 3a), 2492–2506 (oligo 3b), 2438–2452 (oligo 4), 2354–2368 (oligo 4a), 2229–2243 (oligo 5), 2110–2124 (oligo 5a), 2084–2098 (oligo 6), 2050–2064 (oligo 6a), 2018–2032 (oligo 7), 1983–1997 (oligo 7a), 1903–1917 (oligo 8), 1806–1820 (oligo 8a), 1703–1717 (oligo 9), 2570–2591 (oligo 10), 2489–2522 (oligo 11), 2497–2512 (oligo 12), 2448–2468 (oligo 13), 2416–2443 (oligo 14), 2402–2434 (oligo 15), 2385–2408 (oligo 16), 2362–2381 (oligo 17), 2058–2080 (oligo 18), 1951–1980 (oligo 19), 1931–1957 (oligo 20), and 1923–1947 (oligo 21). 50 S-subunit proteins cross-linked to (2N3A76)tRNAPhe were analyzed by two-dimensional PAGE (13Wower J. Hixson S.S. Zimmermann R.A. Biochemistry. 1988; 27: 8114-8121Crossref PubMed Scopus (32) Google Scholar). The labeling of proteins L27 and L33 was established earlier by immunological methods (3Wower J. Hixson S.S. Zimmermann R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5232-5236Crossref PubMed Scopus (96) Google Scholar, 4Wower J. Scheffer P. Sylvers L.A. Wintermeyer W. Zimmermann R.A. EMBO J. 1993; 12: 617-623Crossref PubMed Scopus (39) Google Scholar, 13Wower J. Hixson S.S. Zimmermann R.A. Biochemistry. 1988; 27: 8114-8121Crossref PubMed Scopus (32) Google Scholar). Irradiation of 2-azidoadenosine with near-UV light induces very short cross-links to adjacent molecules and can therefore provide precise information on the molecular environment of the photoreactive base (27Sylvers L.A. Wower J. Bioconjug. Chem. 1993; 4: 411-418Crossref PubMed Scopus (42) Google Scholar). Replacement of the 3′-terminal adenosine of yeast tRNAPhe with 2-azidoadenosine yields a photoreactive derivative, (2N3A76)tRNAPhe, which can be aminoacylated by yeast phenylalanyl-tRNA synthetase and bound toE. coli ribosomes (14Sylvers L. Wower J. Hixson S.S. Zimmermann R.A. FEBS Lett. 1989; 245: 9-13Crossref PubMed Scopus (21) Google Scholar). In the present work, we describe the preparation of 32P-labeled (2N3A76)tRNAPhe derivatives and their incorporation into ribosomal complexes representing different stages in the translation elongation cycle. The photoreactive and non-photoreactive tRNA derivatives usually contained 3H or14C labels in either the aminoacyl moiety or the 3′-terminal nucleotide to verify site occupancy and puromycin reactivity (Table I and “Experimental Procedures”). Identification of the cross-linking sites allowed us to delineate the path followed by the 3′ terminus of the tRNA during its transit through the ribosome.Table ISegments and nucleotides of 23 S rRNA labeled by (2N3A76)tRNA derivatives bound to the A, R, P, and E sites on the E. coli ribosomeA siteP siteE sitePoly(U)Nucleotides labeledRNA segments labeledMajorMinorH segmentsF segmentsT segments(pmol of tRNA/pmol of 70 S)(2N3A)tRNA+U2585U2506H1 > H2F1, F2aT11, T12(0.64)AcPhe-(2N3A)tRNA+U2506G2069,H2 > H1 > H4F1, F2a, F4T11, T12, T18(0.53)U2585AcPhe-(2N3A)tRNA−U2506G2069,H2 > H1 > H4F1, F2a, F2b, F4T11, T12, T13, T18(0.51)C2452, U2585AcPhe-tRNA(2N3A)tRNA+U2585H1F1T11(1.22) 1-aSome tRNA also bound to other sites.(0.59)Phe-(2N3A)tRNAtRNA+U2585U2506H1 > H2F1, F2aT11, T12(0.43)(1.10) 1-aSome tRNA also bound to other sites.Phe-(2N3A)tRNAtRNA+U2585U1926,H1 > H2 > H5F1, F2a, F5T11, T12, T19, T20(0.41) 1-bPhe-tRNA bound to ribosomal A/T or R site as ternary complex with EF-Tu and GMPPNP.(0.89)U2506AcPhe-(2N3A)tRNAtRNA+U2506U2585H2 > H1F1, F2aT11, T12(0.33)(0.53)AcPhe-tRNAAcPhe-tRNA(2N3A)tRNA+C2422H3F3T14, T15(1.41)(0.15)(2N3A)tRNA(2N3A)tRNA(2N3A)tRNA+C2422,U1926,H1–H5F1–F5T11–T20(2.38)U2506, U2585G2069Binding data represent averages calculated from three to five independent cross-linking experiments and are expressed in pmol of tRNA/pmol of 705 ribosomes. Site occupancy was determined from the [3H]Phe-, [14C]Phe- and [14C] labels in nonphotoreactive tRNAs as well as the [32P] label in the photoreactive tRNAs. The relative amounts of cross-linking were inferred from the distribution of radioactivity among H segments by phosphorimaging. The F segment analysis was not quantitative, however, nor was the RNase T1 analysis owing differences in the accessibility of the various segments.1-a Some tRNA also bound to other sites.1-b Phe-tRNA bound to ribosomal A/T or R site as ternary complex with EF-Tu and GMPPNP. Open table in a new tab Binding data represent averages calculated from three to five independent cross-linking experiments and are expressed in pmol of tRNA/pmol of 705 ribosomes. Site occupancy was determined from the [3H]Phe-, [14C]Phe- and [14C] labels in nonphotoreactive tRNAs as well as the [32P] label in the photoreactive tRNAs. The relative amounts of cross-linking were inferred from the distribution of radioactivity among H segments by phosphorimaging. The F segment analysis was not quantitative, however, nor was the RNase T1 analysis owing differences in the accessibility of the various segments. To simulate P-site complexes containing tRNA in the pre- and post-transpeptidation states of binding, Ac[14C]Phe-[5′-32P](2N3A76)tRNAPheand [5′-32P](2N3A76)tRNAPhe were individually bound to poly(U)-programmed 70 S ribosomes (Fig. 1,a and b). Occupancy of the P site by Ac[14C]Phe-[5′-32P](2N3A76)tRNAPhewas confirmed by its quantitative reaction with puromycin. For EF-Tu·GTP-dependent binding to the A site, [14C]Phe-[5′-32P](2N3A76)tRNAPhewas bound to poly(U)-programmed 70 S ribosomes in which the P site was blocked by deacylated E. coli[14C]tRNAPhe. To retain [14C]Phe-[5′-32P](2N3A76)tRNAPhein the R or A/T site (2Moazed D. Noller H.F. Nature. 1989; 342: 142-148Crossref PubMed Scopus (608) Google Scholar, 28Lake J.A. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 1903-1907Crossref PubMed Scopus (102) Google Scholar), GTP was replaced with its non-hydrolyzable analogue, GMPPNP (see Fig. 1 c). During the elongation phase of protein synthesis, the ribosome oscillates between the pre- and post-translocational states. Pre-translocational complexes were formed by the non-enzymatic binding of Ac[14C]Phe-[5′-32P](2N3A76)tRNAPheto the A site of poly(U)-programmed 70 S ribosomes in which the P site was filled with deacylated E. coli[14C]tRNAPhe (see Fig. 1 d) or of Ac[3H]Phe-tRNAPhe to the A site of poly(U)-programmed 70 S ribosomes in which the P site was filled with deacylated [5′-32P](2N3A76)tRNAPhe (see Fig.1 e). Comparison of the latter state with that depicted in Fig. 1 a was expected to show whether the binding of tRNA to the A site influences the arrangement of tRNA in the P site. E site complexes were formed by binding deacylated [5′-32P](2N3A76)tRNAPhe to poly(U)-programmed ribosomes under conditions in which both the A and P sites were blocked with Ac[14C]Phe-tRNAPhe(see Fig. 1 f) (29Kirillov S.V. Semenkov Yu. P. FEBS Lett. 1982; 148: 235-238Crossref PubMed Scopus (34) Google Scholar). The finding that poly(U)-programmed E. coli ribosomes can bind three molecules of deacylated tRNAPhe in vitro was crucial for the development of the three-site model of the ribosome, which suggests that tRNAs bind successively to the A, P, and E sites during the elongation cycle (30Kirillov S.V. Makarov E.M. Semenkov Yu. P. FEBS Lett. 1983; 157: 91-94Crossref PubMed Scopus (85) Google Scholar, 31Lill R. Robertson J.M. Wintermeyer W. Biochemistry. 1984; 23: 6710-6717Crossref PubMed Scopus (77) Google Scholar, 32Rheinberger H.J. Sternbach H. Nierhaus K.H. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5310-5314Crossref PubMed Scopus (162) Google Scholar, 33Grajevskaja R.A. Ivanov Y.V. Saminsky E.M. Eur. J. Biochem. 1982; 128: 47-52Crossref PubMed Scopus (83) Google Scholar). Therefore, we examined the pattern of cross-linking in the presence of saturating amounts of deacylated [5′-32P](2N3A76)tRNAPhe in addition to the complexes described above (Fig. 1 g). Each tRNA-ribosome complex was irradiated with 300-nm lamps to induce cross-linking (3Wower J. Hixson S.S. Zimmermann R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5232-5236Crossref PubMed Scopus (96) Google Scholar). Separation of the tRNA-ribosome complexes into 30 S and 50 S subunit fractions by centrifugation at low Mg2+concentration demonstrated that the tRNA derivatives cross-linked exclusively to the 50 S subunit. Subsequent analysis of the covalent tRNA-50 S subunit complexes on a sucrose gradient containing SDS showed that the cross-links are distributed between the 23 S rRNA and the 50 S subunit proteins. As we have previously reported (3Wower J. Hixson S.S. Zimmermann R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5232-5236Crossref PubMed Scopus (96) Google Scholar, 4Wower J. Scheffer P. Sylvers L.A. Wintermeyer W. Zimmermann R.A. EMBO J. 1993; 12: 617-623Crossref PubMed Scopus (39) Google Scholar, 34Wower J. Wower I.K. Kirillov S. Rosen K. Hixson S.S. Zimmermann R.A. Biochem. Cell Biol. 1995; 73: 1041-1047Crossref PubMed Scopus (32) Google Scholar, 35Wower J. Wower I.K. Zwieb C.W. Nucleic Acids Symp. 1999; 41: 187-191Google Scholar), A and P site-bound (2N3A76)tRNAPhe and its aminoacylated derivatives primarily label protein L27, whereas E site-bound (2N3A76)tRNAPhe labels proteins L33 and L1. The sites to which (2N3A76)tRNAPhecross-links on the 23 S rRNA were determined by a combination of three approaches, RNase H cleavage, RNase protection, and primer extension, as outlined in Fig. 2. Covalent [5′-32P](2N3A76)tRNAPhe-23 S rRNA complexes were digested with RNase H in the presence of pairs of 15-mer oligodeoxyribonucleotides complementary to sequences located approximately 100–250 nucleotides apart in the primary structure of the 23 S rRNA (for a list of oligonucleotides see “Experimental Procedures”). Cleavage of the rRNA at sites that bracket the cross-link releases a fragment, which is tagged by covalently attached [5′-32P](2N3A76)tRNAPhe or its derivatives and can be readily detected by denaturing PAGE (Fig. 1). Such “scans” of the covalent tRNA-23 S rRNA complexes revealed five labeled segments within domains IV and V of the 23 S rRNA, which encompass nucleotides 2567–2904 (segment H1), 2446–2568 (segment H2), 2237–2445 (segment H3), 2025–2092 (segment H4), and 1910–2024 (segment H5). The specific pattern of cross-linking depended on the conditions under which the photoreactive tRNAs were bound to the ribosome (Fig. 1). To delineate the sites of cross-linking more narrowly, [5′-32P](2N3A76)tRNAPhe-23 S rRNA complexes were subjected to further RNase H digestions using pairs of oligodeoxynucleotides that differed from those used in the initial scan. Products of these digestions, denoted F fragments, were fractionated by denaturing PAGE (Figs. 2 and3 a). This step was followed by RNase protection analysis in which complementary oligodeoxynucleotides were hybridized to the F regions of the 23 S rRNA and the resulting heteroduplexes were digested with RNase T1. Protected fragments that retained the 32P label derived from cross-linked [5′-32P](2N3A76)tRNAPhe moieties, designated T fragments, delimited the sites of cross-linking to sequences of the 23 S RNA ranging from 10 to 20 nucleotides in length (Figs. 2 and 3 b). Final identification of the cross-linking sites was carried out using the primer extension method (26Moazed D. Stern S. Noller H.F. J. Mol. Biol. 1986; 187: 399-416Crossref PubMed Scopus (447) Google Scholar). For this purpose, fragments of approximately 250 nucleotides spanning each of the five cross-linking sites in the 23 S rRNA were excised from the appropriate [5′-32P](2N3A76)tRNAPhe-23 S rRNA complexes with RNase H, separated by polyacrylamide gel electrophoresis as in Fig. 1 and subjected to primer extension analysis (Fig. 3,c and d). When tRNA-23 S rRNA complexes containing tRNA cross-links to H1 were digested with RNase H in the presence of oligonucleotides 2, 2a, and 3, all of which resulted in the excision of 3′-terminal segments of 23 S rRNA, only the longest was tagged by [5′-32P](2N3A76)tRNAPhe (Figs. 2and 3 a). The cross-link site must therefore be located within fragment F1, which encompasses nucleotides 2567–2590. RNase T1 protection analysis showed that oligonucleotide 10 protected [5′-32P](2N3A76)tRNAPhe-tagged subfragment T10, which corresponds to nucleotides 2570–2592 (Figs. 2and 3 b). The only new primer extension stop within this sequence is at U2586 (Figs. 3 c and4), one nucleotide before the cross-link site. Covalent tRNA-23 S rRNA complexes, in which [5′-32P](2N3A76)tRNAPhelabeled segment H2, were cleaved with RNase H in the presence of oligonucleotides 3, 3a, 3b, and 4. Inspection of the radioactively labeled sequences released in this assay revealed that the cross-link site is located within fragment F2a, which spans nucleotides 2499–2539 (Fig. 2). RNase T1 protection experiments revealed that subfragment T11, which corresponds to nucleotides 2488–2524, retained the [5′-32P](2N3A76)tRNAPhe tag (Figs. 2 and 3 b). Primer extension analysis identified the cross-linked nucleotide as U2506 (Fig. 3 d). When a similar analysis was carried out on tRNA-23 S rRNA complexes prepared from ribosomes in the absence of poly(U), two sites of cross-linking were identified within segment H2. One of them was located within fragment F2a and corresponded to U2506, whereas the other was found to be in fragment F2b, which encompasses nucleotides 2446–2498 (Fig. 2). The site of attachment within F2b was determined by primer extension to be C2452. RNase H cleavage of all tRNA-23 S rRNA complexes in which tRNA was cross-linked to H3 in the presence of the oligonucleotide pairs 4/4a and 4a/5 demonstrated that the site of cross-linking is located in fragment F3, which encompasses nucleotides 2360–2445 (Fig. 2). This portion of the complex was further analyzed by RNase T1 protection using oligonucleotides 14, 15, 16, and 17. Because oligonucleotides 14 and 15 protected two overlapping [5′-32P](2N3A76)tRNAPhe-labeled subfragments, denoted T14 and T15 in Fig. 2, the site of cross-linking must be located between nucleotides 2415 and 2436. The nucleotide covalently attached to the tRNA was determined to be C2422 by primer extension analysis. RNase H digestion of tRNA-23 S rRNA complexes containing tRNA cross-linked to segment H4 in the presence of oligonucleotides pairs 5/6, 5/6a, 5/7, and 6a/7a showed that the cross-link is located in fragment F4, between nucleotides 2024 and 2091 (Fig. 2). RNase T1 protection experiments utilizing oligonucleotide 18 localized the cross-linking site to the sequence encompassing nucleotides 2058–2083 (Fig. 2). Primer extension analysis demonstrated that G2069 was the residue covalently linked to tRNA. When oligonucleotide pairs 7/8 and 7a/8a were used to direct RNase H cleavage of tRNA-23 S rRNA complexes containing tRNA cross-linked to segment H5, the site of tRNA attachment was found to be delimited by nucleotides 1910–1990 within fragment F5 (Fig. 2). Hybridization to oligonucleotides 19, 20, and 21 revealed that only oligonucleotide 21 protects the 23 S rRNA region tagged by [5′-32P](2N3A76)tRNAPhe from RNase T1 digestion; the site of cross-linking must therefore be located between nucleotides 1922 and 1929 (see Fig. 2). Unequivocal identification of the cross-linked nucleotide within this sequence proved impossible owing to stops in all lanes at C1924 and A1927. We suggest that the stop at A1927 obscures the cross-link of tRNA to U1926, a highly conserved nucleotide that is protected from chemical modification by P site tRNA (2Moazed D. Noller H.F. Nature. 1989; 342: 142-148Crossref PubMed Scopus (608) Google Scholar). When three deacylated (2N3A76)tRNAPhe molecules were bound simultaneously to poly(U)-programmed ribosomes and then subjected to UV irradiation, cross-links were observed in segments H1, H2, H3, H4, and H5 (Fig. 1 g). Subsequent analysis revealed that the tRNA mainly labeled nucleotides U2506, U2585, and C2422, with smaller amounts cross-linked to nucleotides U1926 and G2069. Because these five nucleotides are exactly the same as those labeled by (2N3A76)tRNAPhe molecules, or their derivatives, placed individually at either the A, P, or E sites, we found no evidence for additional, nonspecific cross-links when tRNA is present in excess. Incorporation of 2-azidoadenosine into tRNA yields a phot" @default.
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• W2078884107 title "Transit of tRNA through the Escherichia coliRibosome" @default.
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• W2078884107 cites W1589944020 @default.
• W2078884107 cites W1688418683 @default.
• W2078884107 cites W1964652224 @default.
• W2078884107 cites W1965690043 @default.
• W2078884107 cites W1967465227 @default.
• W2078884107 cites W1970107043 @default.
• W2078884107 cites W1974432284 @default.
• W2078884107 cites W1978352011 @default.
• W2078884107 cites W1980272310 @default.
• W2078884107 cites W1982247071 @default.
• W2078884107 cites W1983121331 @default.
• W2078884107 cites W1994663143 @default.
• W2078884107 cites W1994954153 @default.
• W2078884107 cites W1995418797 @default.
• W2078884107 cites W2001560423 @default.
• W2078884107 cites W2007874938 @default.
• W2078884107 cites W2008025258 @default.
• W2078884107 cites W2013303604 @default.
• W2078884107 cites W2016001703 @default.
• W2078884107 cites W2017771698 @default.
• W2078884107 cites W2021761289 @default.
• W2078884107 cites W2022082484 @default.
• W2078884107 cites W2035935848 @default.
• W2078884107 cites W2037934177 @default.
• W2078884107 cites W2039846261 @default.
• W2078884107 cites W2044905393 @default.
• W2078884107 cites W2045690036 @default.
• W2078884107 cites W2049193281 @default.
• W2078884107 cites W2053524548 @default.
• W2078884107 cites W2057924122 @default.
• W2078884107 cites W2064521337 @default.
• W2078884107 cites W2069458064 @default.
• W2078884107 cites W2076324526 @default.
• W2078884107 cites W2080354279 @default.
• W2078884107 cites W2083411290 @default.
• W2078884107 cites W2084801405 @default.
• W2078884107 cites W2086557819 @default.
• W2078884107 cites W2089457247 @default.
• W2078884107 cites W2092718501 @default.
• W2078884107 cites W2093091879 @default.
• W2078884107 cites W2123984393 @default.
• W2078884107 cites W2155373804 @default.
• W2078884107 cites W2187477592 @default.
• W2078884107 cites W2324570752 @default.
• W2078884107 cites W2744267847 @default.
• W2078884107 cites W75998983 @default.
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