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• W2044246876 abstract "We investigated interaction of an RNA domain covering the target site of α-sarcin and ricin (sarcin/ricin domain) of Escherichia coli 23 S rRNA with ribosomal proteins. RNA fragments comprising residues 2630–2788 (Tox-1) and residues 2640–2774 (Tox-2) of 23 S rRNA were transcribed in vitroand used to analyze the binding proteins by gel shift and filter binding. Protein L6 bound to both Tox-1 (K d: 0.31 μm) and Tox-2 (K d: 0.18 μm), and L3 bound only to Tox-1 (K d: 0.069 μm) in a solution containing 10 mmMgCl2 and 175 mm KCl at 0 °C. Footprinting studies were performed using the chemical probe dimethyl sulfate on full-length 23 S rRNA. Binding of L6 protected a single base, A-2757, and strongly enhanced reactivity of C-2752. A direct role of A-2757 in the L6 binding was verified by site-directed mutagenesis; replacements of A-2757 with G and C impaired the L6 binding. On the other hand, binding of L3 protected A-2632, A-2634, A-2635, A-2675, A-2726, A-2733, A-2749, and A-2750. Interestingly, binding of L6 and L3 together protected additional bases A-2657, A-2662, C-2666, and C-2667 in the sarcin/ricin loop, in addition to A-2740, A-2741, A-2748, A-2753, A-2764, A-2765, and A-2766 in the other stem-loop. This appears to be due to cooperative interaction of L3 and L6 with the RNA. The results are discussed with respect to conformational modulation of the sarcin/ricin domain by the protein binding. We investigated interaction of an RNA domain covering the target site of α-sarcin and ricin (sarcin/ricin domain) of Escherichia coli 23 S rRNA with ribosomal proteins. RNA fragments comprising residues 2630–2788 (Tox-1) and residues 2640–2774 (Tox-2) of 23 S rRNA were transcribed in vitroand used to analyze the binding proteins by gel shift and filter binding. Protein L6 bound to both Tox-1 (K d: 0.31 μm) and Tox-2 (K d: 0.18 μm), and L3 bound only to Tox-1 (K d: 0.069 μm) in a solution containing 10 mmMgCl2 and 175 mm KCl at 0 °C. Footprinting studies were performed using the chemical probe dimethyl sulfate on full-length 23 S rRNA. Binding of L6 protected a single base, A-2757, and strongly enhanced reactivity of C-2752. A direct role of A-2757 in the L6 binding was verified by site-directed mutagenesis; replacements of A-2757 with G and C impaired the L6 binding. On the other hand, binding of L3 protected A-2632, A-2634, A-2635, A-2675, A-2726, A-2733, A-2749, and A-2750. Interestingly, binding of L6 and L3 together protected additional bases A-2657, A-2662, C-2666, and C-2667 in the sarcin/ricin loop, in addition to A-2740, A-2741, A-2748, A-2753, A-2764, A-2765, and A-2766 in the other stem-loop. This appears to be due to cooperative interaction of L3 and L6 with the RNA. The results are discussed with respect to conformational modulation of the sarcin/ricin domain by the protein binding. It is generally recognized that rRNA plays a fundamental role in translation and associated ribosomal proteins help it by modulating the conformation and dynamics of rRNA (reviewed in Ref. 1Noller H.F. Annu. Rev. Biochem. 1991; 60: 191-227Crossref PubMed Scopus (415) Google Scholar). Detailed knowledge of rRNA-protein interaction is, therefore, important to understand the mechanism of action of rRNA, particularly of the conserved functional regions. The sarcin/ricin loop comprising residues 2653–2667 in domain VI of Escherichia coli 23 S rRNA is one of the most notable regions, since this includes an important site of interaction of elongation factors EF-G 1The abbreviations used are: EF, elongation factor; DMS, dimethyl sulfate. and EF-Tu with the ribosome (2Hausner T.P. Atmadja J. Nierhause K.H. Biochimie (Paris). 1987; 69: 911-923Crossref PubMed Scopus (159) Google Scholar, 3Moazed D. Robertson J.M. Noller H.F. Nature. 1988; 334: 362-364Crossref PubMed Scopus (417) Google Scholar, 4Munishkin A. Wool I.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12280-12284Crossref PubMed Scopus (79) Google Scholar) and also the site of action for ribotoxins, α-sarcin and ricin (5Endo Y. Wool I.G. J. Biol. Chem. 1982; 257: 9054-9060Abstract Full Text PDF PubMed Google Scholar, 6Endo Y. Tsurugi K. J. Biol. Chem. 1988; 263: 8735-8739Abstract Full Text PDF PubMed Google Scholar). It is not clear whether a certain ribosomal protein interacts with this region and affects the conformation in the ribosome, although a model of the three-dimensional structure of this loop region has been built up on the basis of NMR data (7Szewczak A.A. Moore P.B. Chan Y.-L. Wool I.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9581-9585Crossref PubMed Scopus (228) Google Scholar, 8Szewczak A.A. Moore P.B. J. Mol. Biol. 1995; 247: 81-98Crossref PubMed Scopus (187) Google Scholar). There is evidence suggesting that assembly of ribosomal proteins to 23 S rRNA changes a feature of the sarcin/ricin loop region; (a) many sites at or near the loop region in the naked rRNA are much more reactive with a variety of chemicals and ribonucleases than those in the 50 S subunit (9Leffers H. Egebjerg J. Andersen A. Christensen T. Garrett R.A. J. Mol. Biol. 1988; 204: 507-522Crossref PubMed Scopus (53) Google Scholar), and (b) this loop region is accessible to ricin in the naked 23 S rRNA, but not in the intactE. coli ribosome (6Endo Y. Tsurugi K. J. Biol. Chem. 1988; 263: 8735-8739Abstract Full Text PDF PubMed Google Scholar). Leffer et al. (9Leffers H. Egebjerg J. Andersen A. Christensen T. Garrett R.A. J. Mol. Biol. 1988; 204: 507-522Crossref PubMed Scopus (53) Google Scholar) identified proteins L3 and L6 that bound to domain VI comprising residues 2629 to the 3′ end of 23 S rRNA. By a footprinting approach, they localized the major binding site for L3 on the 2630–2644/2771–2788 stem, a root of the large RNA domain including the sarcin/ricin loop region, but failed to determine the L6 binding site (9Leffers H. Egebjerg J. Andersen A. Christensen T. Garrett R.A. J. Mol. Biol. 1988; 204: 507-522Crossref PubMed Scopus (53) Google Scholar). Although the binding affinity of L6 to rRNA is low (9Leffers H. Egebjerg J. Andersen A. Christensen T. Garrett R.A. J. Mol. Biol. 1988; 204: 507-522Crossref PubMed Scopus (53) Google Scholar, 10Spierer P. Zimmermann R.A. Mackie G.A. Eur. J. Biochem. 1975; 52: 459-468Crossref PubMed Scopus (44) Google Scholar), this ability is supported by recent crystallographic data on this protein that show a domain structure homologous with a large family of RNA binding proteins (11Golden B.L. Ramakrishnan V. White S.W. EMBO J. 1993; 12: 4901-4908Crossref PubMed Scopus (66) Google Scholar). Protein L6 is interesting with respect to the location and function in the ribosome. Data on immunoelectron microscopy and protein-protein cross-linking indicate that L6 is located in a position close to the base of L7/L12 stalk and to proteins L10 and L11 (12Stöffler-Meilicke M. Noah M. Stöffler G. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6780-6784Crossref PubMed Scopus (43) Google Scholar, 13Walleczek J. Schüler D. Stöffler-Meilicke M. Brimacombe R. Stöffler G. EMBO J. 1988; 7: 3571-3576Crossref PubMed Scopus (91) Google Scholar, 14Traut R.R. Tewari D.S. Sommer A. Gavino G.R. Olson H.M. Glitz D.G. Hardesty B. Kramer G. Structure, Function and Genetics of Ribosomes. Springer-Verlag, New York1985: 286-308Google Scholar), and this area is mapped out as the binding sites for EF-G (15Girshovich A.S. Kurtskhalia T.V. Ovchinnikov Y.A. Vasiliev V.D. FEBS Lett. 1981; 130: 54-59Crossref PubMed Scopus (58) Google Scholar, 16Agrawal R.K. Penczek P. Grassucci R.A. Frank J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6134-6138Crossref PubMed Scopus (306) Google Scholar) and EF-Tu (17Girshovich A.S. Bochkareva E.S. Vasiliev V.D. FEBS Lett. 1986; 197: 192-198Crossref PubMed Scopus (53) Google Scholar, 18Stark H. Rodnina M.V. Rinke-Appel J. Brimacombe R. Wintermeyer W. van Heel M. Nature. 1997; 389: 403-406Crossref PubMed Scopus (314) Google Scholar) in the ribosome. In fact, L6 cross-links to EF-G (19Sköld S.-E. Eur. J. Biochem. 1982; 127: 225-229Crossref PubMed Scopus (28) Google Scholar). These lines of evidence suggest that L6 is one of the members constructing the factor binding site in the ribosome. We here investigate the interaction of the RNA domain containing the sarcin/ricin loop region of 23 S rRNA (termed here the sarcin/ricin domain) with ribosomal proteins by gel retardation and footprinting, and demonstrate that L6 directly binds to a site within residues 2640–2774 of 23 S rRNA. By footprinting, we also show that L6, together with L3, protects 4 bases in the sarcin/ricin loop against chemical modification. Our data provide valuable information about features of the sarcin/ricin domain in the ribosome. The DNA fragments comprising residues 2630–2788 (Tox-1), and residues 2640–2774 (Tox-2) (see Fig. 4) were amplified using the polymerase chain reaction (20Saiki R.K. Gelfand D.H. Stoffel S. Share S.J. Higuchi R. Hoen G.T. Mullis K.B. Erlich H.A. Science. 1988; 239: 487-491Crossref PubMed Scopus (13561) Google Scholar), and inserted into HindIII andXbaI sites of an expression vector, pSPT18 (Boehringer Mannheim). Base substitutions of A-2757 with G and C in Tox-2 were performed by oligonucleotide-directed mutagenesis in polymerase chain reaction (21Higuchi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2109) Google Scholar) using primers containing the individual mutations. DNA sequences of the obtained constructs were verified by dideoxy sequencing (22Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52928) Google Scholar). The plasmid construction for the RNA fragment containing residues 1029–1127 (corresponds to the GTPase domain) of 23 S rRNA was as described previously (23Uchiumi T. Wada A. Kominami R. J. Biol. Chem. 1995; 270: 29889-29893Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The RNA fragments were synthesized in a solution (200 μl) containing 1,000 units of SP-6 RNA polymerase, 4 μg of template DNA linearized at the XbaI site, 40 mm Tris-HCl, pH 8.0, 14 mmMgCl2, 10 mm NaCl, 5 mmdithiothreitol, 1 mm spermidine, 10 μg of bovine serum albumin, 2 mm each of ATP, GTP, and CTP, and 0.5 mm UTP (supplemented with 50 μCi of [α-32P]UTP). The transcripts were purified by gel filtration on a Sephadex G-50 column (Amersham Pharmacia Biotech). The large ribosomal subunits were prepared from E. coli Q13, as described previously (24Wada A. J. Biochem. (Tokyo). 1986; 100: 1583-1594Crossref PubMed Scopus (78) Google Scholar). Total proteins (TP50) extracted from the subunits (25Hardy S.J.S. Kurland C.G. Voynow P. Mora G. Biochemistry. 1969; 8: 2897-2904Crossref PubMed Scopus (809) Google Scholar) were fractionated by a stepwise elution from CM-cellulose (Whatman) column equilibrated with a buffer (7 m urea, 5 mm 2-mercaptoethanol, and 20 mm sodium acetate, pH 4.6) using increasing concentrations of LiCl (m), 0.04, 0.075, 0.1, 0.15, 0.2, 0.25, and 0.3. A part of each fraction was dialyzed against the renaturation buffer consisting of 0.35m KCl, 5 mm 2-mercaptoethanol, and 20 mm Tris-HCl, pH 7.5, and tested for binding to Tox-1 by gel retardation, as described below. Protein fractions containing the binding activity were further purified by high performance ion-exchange chromatography; the protein fractions were applied to a CM-5PW column (Tosoh) equilibrated with a buffer consisting of 6 m urea, 20 mm sodium phosphate, pH 6.5, 80 mm LiCl, and 5 mm 2-mercaptoethanol, and eluted with a linear gradient of 80–280 mm LiCl. The isolated proteins were concentrated with Centricon-10 (Amicon), dialyzed against the renaturation buffer, and tested for the binding to Tox-1. Identification of the isolated proteins were performed by two-dimensional polyacrylamide gel electrophoresis (24Wada A. J. Biochem. (Tokyo). 1986; 100: 1583-1594Crossref PubMed Scopus (78) Google Scholar) and amino acid sequencing of the N termini (Applied Biosystems). A solution (5 μl) containing 5 pmol of the [32P]RNA fragments, 20 mmMgCl2, 0.35 m KCl, 30 mm Tris-HCl, pH 7.5, was preincubated at 40 °C for 20 min. After addition of 5 μl of 30 mm Tris-HCl, pH 7.5, and a protein sample as indicated in the figure legends, the mixture was incubated further at 30 °C for 10 min. RNA-protein binding was examined by electrophoresis in 6% polyacrylamide gel (acrylamide/bisacrylamide ratio of 40:1) at 6.5 V/cm, with buffer recirculation at 4 °C. The two buffer systems were used in gel analyses; system 1 contained 5 mm MgCl2, 50 mm KCl, and 50 mm Tris-HCl, pH 7.6, and system 2 contained 4 mm MgCl2, and 20 mm Tris-boric acid, pH 7.6. Radiolabeled RNA fragments (5 pmol for L6 binding and 1 pmol for L3 binding) were preincubated at 40 °C for 20 min in 50 μl of 350 mm KCl, 20 mmMgCl2, 30 mm Tris-HCl, pH 7.5. By mixing with 30 mm Tris-HCl, pH 7.5, and a protein sample, the solution was adjusted to 100 μl. The mixture was incubated for another 10 min at 30 °C, and then placed on ice for 10 min. The reaction mixture was filtered through a nitrocellulose membrane (Millipore, type HA, 0.45-μm pore size, 25-mm diameter) as described by Draper et al. (26Draper D.E. Deckman I.C. Vartikar J.V. Methods Enzymol. 1988; 164: 203-220Crossref PubMed Scopus (55) Google Scholar). The filter was counted for 32P using a Beckman LS6000IC Analyzer. A background determined by filtration in the absence of proteins was subtracted from each assay. The data were fitted by nonlinear least-squares analysis with a hyperbolic binding function using Prism 2 (Graphpad Software, San Diego, CA), and theK d values were derived from three experiments. The 23 S rRNA were prepared from the isolated 50 S subunits by phenol extraction and sucrose gradient centrifugation. The 23 S rRNA (20 pmol) was preincubated at 40 °C for 10 min in 25 μl of solution containing 20 mmMgCl2, 350 mm KCl, 50 mm potassium cacodylate, pH 7.2. After adding protein samples, 50 pmol of L3, 100 pmol of L6, or both the proteins, and 50 mm potassium cacodylate, pH 7.2, the solution (50 μl) was incubated at 30 °C for 10 min. Chemical modification was started by addition of DMS (1 μl; 1:4 dilution in ethanol), followed by incubation at 30 °C for 15 min. The modified RNA samples were recovered and used as templates of primer extension, as described by Moazed and Noller (27Moazed D. Noller H.F. Cell. 1986; 47: 985-994Abstract Full Text PDF PubMed Scopus (351) Google Scholar). The used primers were 5′-GGAGAACTCATCTCGGGG-3′ and 5′-GTCGTCGTCTTCAACGTT-3′ complementary to residues 2771–2788 and 2813–2830 of 23 S rRNA, respectively. Synthetic RNA fragments that mimic the local structure of rRNA have been widely employed to investigate RNA-protein interactions (4Munishkin A. Wool I.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12280-12284Crossref PubMed Scopus (79) Google Scholar,9Leffers H. Egebjerg J. Andersen A. Christensen T. Garrett R.A. J. Mol. Biol. 1988; 204: 507-522Crossref PubMed Scopus (53) Google Scholar, 23Uchiumi T. Wada A. Kominami R. J. Biol. Chem. 1995; 270: 29889-29893Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 28Ryan P.C. Lu M. Draper D.E. J. Mol. Biol. 1991; 221: 1257-1268Crossref PubMed Scopus (91) Google Scholar, 29Uchiumi T. Traut R.R. Elkon K. Kominami R. J. Biol. Chem. 1991; 266: 2054-2062Abstract Full Text PDF PubMed Google Scholar). In the present study, we synthesized two RNA fragments covering the sarcin/ricin domain of E. coli 23 S rRNA to investigate the interaction of the RNA region with 50 S ribosomal proteins: Tox-1 comprising residues 1630–1788 and Tox-2 comprising residues 2640–2774 (see Fig. 4). Protein binding to the RNA fragments was assayed by gel retardation for each protein fraction separated with CM-cellulose column and for high performance liquid chromatography-purified proteins (see “Materials and Methods”). Two proteins showed ability to interact with Tox-1. They were identified as L3 and L6 by two-dimensional polyacrylamide gel electrophoresis and amino acid sequencing of the N termini (data not shown). Fig.1 A shows the purity of isolated L3 (lane 2) and L6 (lane 3) samples used for the binding experiments. As shown in Fig. 1 B, L3 strongly bound to Tox-1 (lane 2), but not to Tox-2 (lane 5) or to a control RNA fragment containing residues 1029–1127 covering the GTPase domain (lane 8). L6 protein, on the other hand, showed a weak affinity for both Tox-1 (lane 3) and Tox-2 (lane 6), but not for the GTPase domain (lane 9). This gel condition containing 50 mm KCl (system 1, Fig. 1 B) did not detect the stable L6-RNA complex, but another gel system excluding KCl (system 2, Fig. 1 C) showed the L6-RNA complex as a clear band with Tox-1 (lanes 3 and 4), and Tox-2 (lanes 7 and 8) depending on concentrations of added L6. No complex was detected between L6 and the GTPase domain even in system 2 (see lanes 10–12). Binding affinities of L3 and L6 for the RNAs were examined by filter binding assay (Fig. 2). The RNA fragments Tox-1, Tox-2, and GTPase domain were titrated with proteins L3 (Fig.2 A) and L6 (Fig. 2 B). L3 bound to Tox-1 with a high affinity (K d: 0.069 ± 0.025 μm), whereas L6 bound to Tox-1 and Tox-2 with lower affinities (K d: 0.31 ± 0.10 μmand 0.18 ± 0.06 μm, respectively). There was no appreciable binding either of L3 to Tox-2 or the GTPase domain or of L6 to the GTPase domain. These binding data suggest that the primary binding site for L6 lies within Tox-2 and that for L3 in the 2630–2639/2775–2788 region, which remains in Tox-1 and is deleted in Tox-2. Binding sites for L3 and L6 in 23 S rRNA were further analyzed by DMS-footprinting. Sites of the chemical modification (at N-1 of adenine and N-3 of cytosine) in the presence or absence of proteins were localized by primer-extension, followed by gel electrophoresis (Fig.3). Binding of L3 protected bases A-2632, A-2634, A-2635, A-2675, A-2726, A-2733, A-2749, and A-2750 and enhanced the modification at A-2734 (lane 2). These bases lie in the 2630–2643/2771–2788 stem, the 2675–2733 stem, and the 2747–2757 loop (Fig. 4). Binding of L6 caused marked protection at A-2757 and enhancement at C-2752 (Fig. 3,lane 3) within the conserved loop region of residues 2747–2757 (Fig. 4). L6 also enhanced C-2699 weakly, but obviously. No effect was observed in the sarcin/ricin loop comprising residues 2653–2667 by the individual bindings. However, additions of L3 and L6 together newly caused strong protection of 4 bases in this loop region, A-2657, A-2662, C-2666, and C-2667, in addition to A-2740, A-2741, A-2748, A-2753, A-2764, A-2765, and A-2766 in the stem-loop of residues 2735–2769 (Figs. 3, lane 4, and 4). We infer that these effects given by the combination of L3 and L6 reflect a cooperative interaction of the two proteins with the RNA. Despite the physical binding of L6 to Tox-2 (Figs. 1 and 2), our DMS-footprinting data showed protection of only A-2757 in this region (Fig. 3). To clarify whether A-2757 is involved in L6 binding, we performed a site-directed mutagenesis. Effect of base substitutions of A-2757 with G and C on L6 binding to Tox-2 was tested by gel retardation in system 2. As shown in Fig.5, the binding was disrupted by either A to C transversion (lane 4) or A to G transition (lane 6). These results combined with the footprinting data suggest that A-2757 plays a direct role in L6 binding to the RNA. Little has been known about ribosomal proteins assembled to an RNA domain including the conserved sarcin/ricin loop region. The present study demonstrates that protein L6 directly binds to a limited region within residues 2640–2774 (Tox-2). The marked effects of L6 binding on DMS modification is localized in the conserved loop of residues 2747–2757: protection of A-2757 and enhancement of C-2752 (Fig. 4). In addition, replacement of this protected base A-2757 with G or C causes disruption of L6 binding (Fig. 5). The results suggest that L6 recognizes a local structure including A-2757 within Tox-2 as a primary binding site. The present results are in line with previous data on RNA binding ability of L6 to the 3′ half (10Spierer P. Zimmermann R.A. Mackie G.A. Eur. J. Biochem. 1975; 52: 459-468Crossref PubMed Scopus (44) Google Scholar) and to domain VI of 23 S rRNA (9Leffers H. Egebjerg J. Andersen A. Christensen T. Garrett R.A. J. Mol. Biol. 1988; 204: 507-522Crossref PubMed Scopus (53) Google Scholar), and also consistent with cross-linking between a mammalian homologue (L9) of E. coli L6 and ricin A chain, which attacks the sarcin/ricin loop (30Vater C.A. Bartle L.M. Leszyk J.D. Lambert J.M. Goldmacher V.S. J. Biol. Chem. 1995; 270: 12933-12940Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). On the other hand, L6 is cross-linked to a region of residues 2473–2481 of domain V of 23 S rRNA with 2-iminothiolane (31Wower I. Wower J. Meinke M. Brimacombe R. Nucleic Acids Res. 1981; 9: 4285-4302Crossref PubMed Scopus (61) Google Scholar), although direct binding of L6 to an RNA fragment for domain V was not detected (9Leffers H. Egebjerg J. Andersen A. Christensen T. Garrett R.A. J. Mol. Biol. 1988; 204: 507-522Crossref PubMed Scopus (53) Google Scholar). It is presumed that the 2473–2481 region of domain V lies in the close proximity to, if not directly interacts with, L6 in the ribosome. Another protein, L3, strongly binds to Tox-1, but not to Tox-2, suggesting that the region of residues 2630–2639/2775–2788 lacking in Tox-2 is the major binding site for L3. This is consistent with previous footprinting data by Leffers et al. (9Leffers H. Egebjerg J. Andersen A. Christensen T. Garrett R.A. J. Mol. Biol. 1988; 204: 507-522Crossref PubMed Scopus (53) Google Scholar). In our footprinting study, L3 protects not only 3 bases in the 2630–2639/2775–2788 region, but also 5 bases in Tox-2 region (Fig.4). Therefore, together with the 2630–2639/2775–2788 region, some parts within Tox-2 may constitute L3 binding site. Interestingly, our footprinting data also reveal additional protections at the sarcin/ricin loop by the combination of L3 and L6; bases A-2657, A-2662, C-2666, and C-2667 in the sarcin/ricin loop in addition to 7 bases in another stem-loop of residues 2735–2769 are protected by binding of L3 and L6 together (Fig. 4). All of these positions are not reactive with DMS within the 50 S subunit (data not shown), suggesting that the present data reflect the structural feature of the sarcin/ricin domain in the intact ribosome. It is not clear, however, at present whether these additional protections are caused by contact with protein(s) or conformational change of the RNA induced by the two proteins. L3 is known as one of the important proteins bound earliest during the subunit assembly (32Nowotny V. Nierhaus K.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7238-7242Crossref PubMed Scopus (78) Google Scholar) and expected to induce a conformational change of 23 S rRNA, which leads to the next step of the 50 S subunit assembly. Therefore, it is most likely that L3 binding affects a conformation of the Tox-2 region, although there is no detectable effect of L3 alone on the DMS modification at the sarcin/ricin loop. From these considerations, we deduce a tentative model of the RNA folding: the 2630–2644/2771–2788 stem and the 2675–2732 stem fold close together in one side by the strong L3 binding, and in the other side, the sarcin/ricin loop region and the 2735–2769 stem-loop region become closer as much as L6 interacts with both regions and protects them. Further extensive investigations are required to elucidate the mechanism of this cooperative interaction of L3 and L6 with the RNA. The position of L6 near the base of the L7/L12 stalk in the 50 S ribosomal subunit has been established from immunoelectron microscopy (12Stöffler-Meilicke M. Noah M. Stöffler G. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6780-6784Crossref PubMed Scopus (43) Google Scholar, 13Walleczek J. Schüler D. Stöffler-Meilicke M. Brimacombe R. Stöffler G. EMBO J. 1988; 7: 3571-3576Crossref PubMed Scopus (91) Google Scholar) and protein-protein cross-linking studies (14Traut R.R. Tewari D.S. Sommer A. Gavino G.R. Olson H.M. Glitz D.G. Hardesty B. Kramer G. Structure, Function and Genetics of Ribosomes. Springer-Verlag, New York1985: 286-308Google Scholar). In addition, this area underneath the L7/L12 stalk has been observed as the binding site for EF-G (15Girshovich A.S. Kurtskhalia T.V. Ovchinnikov Y.A. Vasiliev V.D. FEBS Lett. 1981; 130: 54-59Crossref PubMed Scopus (58) Google Scholar, 16Agrawal R.K. Penczek P. Grassucci R.A. Frank J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6134-6138Crossref PubMed Scopus (306) Google Scholar) and EF-Tu (17Girshovich A.S. Bochkareva E.S. Vasiliev V.D. FEBS Lett. 1986; 197: 192-198Crossref PubMed Scopus (53) Google Scholar, 18Stark H. Rodnina M.V. Rinke-Appel J. Brimacombe R. Wintermeyer W. van Heel M. Nature. 1997; 389: 403-406Crossref PubMed Scopus (314) Google Scholar). The present results demonstrate that the sarcin/ricin RNA domain (Tox-2) of residues 2640–2774 in 23 S rRNA interacts directly with L6, implying that this RNA domain is situated in the vicinity of L6 at the factor binding site for EF-G and EF-Tu in the ribosome. This is consistent with the facts that EF-G is cross-linked to L6 (19Sköld S.-E. Eur. J. Biochem. 1982; 127: 225-229Crossref PubMed Scopus (28) Google Scholar) and that both of EF-G and EF-Tu protects the sarcin/ricin loop from chemical modification (3Moazed D. Robertson J.M. Noller H.F. Nature. 1988; 334: 362-364Crossref PubMed Scopus (417) Google Scholar). Besides the sarcin/ricin domain, another conserved RNA domain termed the GTPase domain (or thiostrepton binding site) in domain II of 23 S rRNA (33Schmidt F.J. Thompson J. Lee K. Dijk J. Cundliffe E. J. Biol. Chem. 1981; 256: 12301-12305Abstract Full Text PDF PubMed Google Scholar, 34Beauclerk A.A.D. Cundliffe E. Dijk J. J. Biol. Chem. 1984; 259: 6559-6563Abstract Full Text PDF PubMed Google Scholar, 35Egebjerg J. Douthwaite S. Liljas A. Garrett R.A. J. Mol. Biol. 1990; 213: 275-288Crossref PubMed Scopus (123) Google Scholar, 36Rosendahl G. Douthwaite S. J. Mol. Biol. 1993; 234: 1013-1020Crossref PubMed Scopus (74) Google Scholar) is known to interact with EF-G (3Moazed D. Robertson J.M. Noller H.F. Nature. 1988; 334: 362-364Crossref PubMed Scopus (417) Google Scholar, 4Munishkin A. Wool I.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12280-12284Crossref PubMed Scopus (79) Google Scholar). These two RNA domains appear to collaborate in EF-G-dependent process of translation, although the two domains are distant in the primary and secondary structure of 23 S rRNA. It is of interest to see the topographical relationship between the sarcin/ricin domain and the GTPase domain on a model of protein topography (Fig.6). Since the sarcin/ricin domain is situated near L6 (present study) and the GTPase domain on L11 (28Ryan P.C. Lu M. Draper D.E. J. Mol. Biol. 1991; 221: 1257-1268Crossref PubMed Scopus (91) Google Scholar, 33Schmidt F.J. Thompson J. Lee K. Dijk J. Cundliffe E. J. Biol. Chem. 1981; 256: 12301-12305Abstract Full Text PDF PubMed Google Scholar), the two RNA domains are expected to be adjacent to each other. This view is strongly supported by recent evidence from directed hydroxyl radical probing for EF-G binding site (37Wilson K.S. Noller H. Cell. 1998; 92: 131-139Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), i.e. both the sarcin/ricin domain and the GTPase domain are cleaved by the same EF-G conjugated with Fe (II) at position 650 of the molecule. The vicinity of these two RNA domains may explain the fact that binding of thiostrepton to the GTPase domain inhibits accessibility of the sarcin/ricin loop for α-sarcin (38Miller S.P. Bodley J.W. Nucleic Acids Res. 1991; 19: 1657-1660Crossref PubMed Scopus (17) Google Scholar). We thank Dr. R. Kominami (Niigata University) for helpful discussion and critical reading of the manuscript and Dr. S. Odani (Niigata University) for helpful advice on the binding experiments." @default.
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