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• W2023798478 abstract "Ribosomal (r) RNAs play a crucial role in the fundamental structure and function of the ribosome. Helix 69 (H69) (position 1906-1924), a highly conserved stem-loop in domain IV of the 23 S rRNA of bacterial 50 S subunits, is located on the surface for intersubunit association with the 30 S subunit by connecting with helix 44 of 16 S rRNA with the bridge B2a. H69 directly interacts with A/T-, A-, and P-site tRNAs during each translation step. To investigate the functional importance of the highly conserved loop sequence (1912-1918) of H69, we employed a genetic method that we named SSER (systematic selection of functional sequences by enforced replacement). This method allowed us to identify and select from the randomized loop sequences of H69 in Escherichia coli 23 S rRNA functional sequences that are absolutely required for ribosomal function. From a library consisting of 16,384 sequence variations, 13 functional variants were obtained. A1912 and U(Ψ)1917 were selected as essential residues in all variants. An E. coli strain having 23 S rRNA with a U to A mutation at position 1915 showed a severe growth phenotype and low translational fidelity. The result could be explained by the fact that the A1915-ribosome variant has weak subunit association, weak A-site tRNA binding, and decreased translational activity. This study proposes that H69 plays an important role in the control of translational fidelity by modulating A-site tRNA binding during the decoding process. Ribosomal (r) RNAs play a crucial role in the fundamental structure and function of the ribosome. Helix 69 (H69) (position 1906-1924), a highly conserved stem-loop in domain IV of the 23 S rRNA of bacterial 50 S subunits, is located on the surface for intersubunit association with the 30 S subunit by connecting with helix 44 of 16 S rRNA with the bridge B2a. H69 directly interacts with A/T-, A-, and P-site tRNAs during each translation step. To investigate the functional importance of the highly conserved loop sequence (1912-1918) of H69, we employed a genetic method that we named SSER (systematic selection of functional sequences by enforced replacement). This method allowed us to identify and select from the randomized loop sequences of H69 in Escherichia coli 23 S rRNA functional sequences that are absolutely required for ribosomal function. From a library consisting of 16,384 sequence variations, 13 functional variants were obtained. A1912 and U(Ψ)1917 were selected as essential residues in all variants. An E. coli strain having 23 S rRNA with a U to A mutation at position 1915 showed a severe growth phenotype and low translational fidelity. The result could be explained by the fact that the A1915-ribosome variant has weak subunit association, weak A-site tRNA binding, and decreased translational activity. This study proposes that H69 plays an important role in the control of translational fidelity by modulating A-site tRNA binding during the decoding process. Ribosomes translate the genetic information contained in mRNAs into proteins. The large (50 S) subunit of the ribosome catalyzes the formation of a peptide bond between the aminoacyl-tRNA (aa-tRNA) 3The abbreviations used are: aa-tRNA, aminoacyl-tRNA; SSER, systematic selection of functional sequences by enforced replacement; CMC, N-cyclohexyl-N′-β-(4-methylmorpholinium)ethylcarbodiimide p-tosylate); SDG, sucrose density gradient; TC, tight-coupled ribosome; Ψ, pseudouridine; m3Ψ, 3-methylpseudouridine; EF, elongation factor; H69, helix 69; Spc, spectinomycin; Amp, ampicillin; Km, kanamycin; Bicine, N,N-bis(2-hydroxyethyl)glycine. bound to the A-site and the peptidyl-tRNA at the P-site. This peptide bond formation takes place at the peptidyltransferase center of the 50 S subunit. Codon-anticodon pairing occurs at the decoding center of the small (30 S) subunit. The aa-tRNA is delivered to the ribosome as a ternary complex of aa-tRNA, EF-Tu, and GTP. Cognate codon recognition is strictly monitored by 16 S rRNA and triggers GTP hydrolysis and dissociation of EF-Tu. This allows aa-tRNA to be accommodated by the A site of the 50 S subunit. Thus, accuracy of protein synthesis is based on the synergistic interplay of the large and small subunits of the ribosome. However, mechanistic insights into the ribosome dynamics during decoding are still rudimentary. The intersubunit bridges of the ribosome are functional sites that are not only necessary for subunit connection but also play roles in translation. Helix 69 (H69) (position 1906-1924) is a highly conserved stem-loop in domain IV of 23 S rRNA of the bacterial 50 S subunit (Fig. 1A). In fact, each base in the loop of H69 shows more than 98% conservation in 436 bacterial rRNAs (www.rna.icmb.utexas.edu). Crystallographic studies revealed that H69 is located on the surface involved in intersubunit association with the 30 S subunit by connecting with helix 44 (h44) of 16 S rRNA forming the bridge B2a; A1912, A1913, A1914, A1918 and A1919 in H69 make contact with positions 1407-1410 and 1494-1495 in h44 and G1517 in h45 (1Yusupov M.M. Yusupova G.Z. Baucom A. Lieberman K. Earnest T.N. Cate J.H. Noller H.F. Science. 2001; 292: 883-896Crossref PubMed Scopus (1672) Google Scholar, 2Schuwirth B.S. Borovinskaya M.A. Hau C.W. Zhang W. Vila-Sanjurjo A. Holton J.M. Cate J.H. Science. 2005; 310: 827-834Crossref PubMed Scopus (1101) Google Scholar) (see Fig. 7A). In the free 50 S subunit, H69 makes a compact structure and interacts with H71 of 23 S rRNA (3Harms J. Schluenzen F. Zarivach R. Bashan A. Gat S. Agmon I. Bartels H. Franceschi F. Yonath A. Cell. 2001; 107: 679-688Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar), whereas in the 70 S ribosome H69 stretches toward the small subunit and interacts with h44 of 16 S rRNA (1Yusupov M.M. Yusupova G.Z. Baucom A. Lieberman K. Earnest T.N. Cate J.H. Noller H.F. Science. 2001; 292: 883-896Crossref PubMed Scopus (1672) Google Scholar). The tip of H69 moves about 13.5 Å during this structural change. In addition, H69 directly interacts with A/T-, A-, and P-site tRNAs during each translation step (1Yusupov M.M. Yusupova G.Z. Baucom A. Lieberman K. Earnest T.N. Cate J.H. Noller H.F. Science. 2001; 292: 883-896Crossref PubMed Scopus (1672) Google Scholar). During the decoding step, aa-tRNA is brought into the A/T-site as a complex with EF-Tu/GTP (ternary complex). Cryoelectron microscopy analyses showed a kinked conformation for aa-tRNA at the A/T state (molecular spring) (4Valle M. Zavialov A. Li W. Stagg S.M. Sengupta J. Nielsen R.C. Nissen P. Harvey S.C. Ehrenberg M. Frank J. Nat. Struct. Biol. 2003; 10: 899-906Crossref PubMed Scopus (293) Google Scholar, 5Valle M. Sengupta J. Swami N.K. Grassucci R.A. Burkhardt N. Nierhaus K.H. Agrawal R.K. Frank J. EMBO J. 2002; 21: 3557-3567Crossref PubMed Scopus (267) Google Scholar). The tip of H69 makes a contact with the hinge of the kink between D- and anticodon-stems in tRNA. This interaction is supposed to facilitate the structural distortion of tRNA that enables the anticodon-stem to fit into the decoding center of 16 S rRNA. In the crystal structure of the 70 S ribosome complexed to both A- and P-site tRNAs, H69 is positioned between the two tRNAs (1Yusupov M.M. Yusupova G.Z. Baucom A. Lieberman K. Earnest T.N. Cate J.H. Noller H.F. Science. 2001; 292: 883-896Crossref PubMed Scopus (1672) Google Scholar). The minor groove of H69 (positions 1908-1909, 1922-1923) interacts with the minor groove of the D-stem of the P-site tRNA (positions 12-13, 25-26) (Fig. 1B), whereas the conserved loop (positions 1913-1915) of H69 makes a contact with the D-stem of A-site tRNA (positions 11-12 and 25-26) (Fig. 1B). Moreover, it has been reported that H69 interacts with various translational factors. In the post-translocational state, H69 is proximal to EF-G (6Agrawal R.K. Spahn C.M. Penczek P. Grassucci R.A. Nierhaus K.H. Frank J. J. Cell Biol. 2000; 150: 447-460Crossref PubMed Scopus (140) Google Scholar). Chemical footprinting analysis revealed that the C-terminal domain (CTD) of IF3 binds to h44 in 16 S rRNA, which is the site of bridge B2a, indicating that CTD of IF3 mimics H69 to protect subunit association during translation initiation (7Dallas A. Noller H.F. Mol. Cell. 2001; 8: 855-864Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Crystallographic and cryoelectron microscopy studies showed that H69 also interacts with RF2 (8Klaholz B.P. Pape T. Zavialov A.V. Myasnikov A.G. Orlova E.V. Vestergaard B. Ehrenberg M. van Heel M. Nature. 2003; 421: 90-94Crossref PubMed Scopus (174) Google Scholar, 9Rawat U.B. Zavialov A.V. Sengupta J. Valle M. Grassucci R.A. Linde J. Vestergaard B. Ehrenberg M. Frank J. Nature. 2003; 421: 87-90Crossref PubMed Scopus (211) Google Scholar), RF3 (10Klaholz B.P. Myasnikov A.G. Van Heel M. Nature. 2004; 427: 862-865Crossref PubMed Scopus (114) Google Scholar), RRF (11Wilson D.N. Schluenzen F. Harms J.M. Yoshida T. Ohkubo T. Albrecht R. Buerger J. Kobayashi Y. Fucini P. EMBO J. 2005; 24: 251-260Crossref PubMed Scopus (99) Google Scholar), and SmpB (12Valle M. Gillet R. Kaur S. Henne A. Ramakrishnan V. Frank J. Science. 2003; 300: 127-130Crossref PubMed Scopus (127) Google Scholar) bound to transfer-messenger RNA-EF-Tu complex. These observations indicate a pivotal role for H69 in various steps during translation.FIGURE 7Molecular interactions in bridge B2a in E. coli 70 S ribosome. A, structure of the H69 loop interacting with h44 and h45. Genetically selected essential bases (A1912 and Ψ1917) are shown in red. R1918 is shown in orange. Other residues in H69 loop are shown in blue. Residues in h44 and h45 of 16 S rRNA involved in bridge B2a are shown in gray. H-bonds are presented by dashed lines. Coordinates were obtained from 2AWB and 2AW7. B, essential A-minor interaction in bridge B2a between H69 and h44.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To more precisely define the functional importance of the highly conserved loop sequence (1912-1918) of H69, we employed a systematic genetic method that we named SSER (systematic selection of functional sequences by enforced replacement). 4N. S. Sato, N. Hirabayashi, and T. Suzuki, submitted for publication. This method allowed us to identify the residues absolutely essential for ribosomal function in Escherichia coli cells from a randomized rRNA library. The library was constructed by completely randomizing the loop sequence of H69 (Fig. 1B). The variants were then subjected to selection, and the selected variants were sequenced. The selected variants contained natural rRNA sequences from other organisms as well as unnatural but nonetheless functional sequences. The results throw new light on the nature of the bases required for H69 function. Biochemical and genetic analysis of H69 variants provide insights into the functional roles played by H69 in translation. Bacterial Strains, Plasmids, and Cultivation—E. coli Δ7rrn strain TA542 (ΔrrnE ΔrrnB ΔrrnA ΔrrnH ΔrrnG::cat ΔrrnC::cat ΔrrnD::cat ΔrecA56/pTRNA66 pHKrrnC) (14Asai T. Zaporojets D. Squires C. Squires C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1971-1976Crossref PubMed Scopus (226) Google Scholar) was kindly provided by Dr. Catherine L. Squires (Tufts University). The rescue plasmid pRB1014 was constructed by introducing the sacB gene and rrnB operon into pMW118 (Ampr) (Nippon gene). The plasmid pRB1024 was constructed from pMW218 (Kmr) (Nippon gene) by inserting the rrnB operon only. The plasmid pHKrrnC in strain TA542 was replaced by pRB101 to generate strain NT101, which was used as the host cell for SSER selection and B-scan analysis. Cells were grown at 37 °C in 2× Luria-Bertani medium. For solid medium, 1.5% agar was added to LB medium. Antibiotics were added at the following concentrations when required: 40 μg/ml spectinomycin (Spc), 100 μg/ml ampicillin (Amp), and 50 μg/ml kanamycin (Km). To induce plasmid replacement in NT101, 5% sucrose was added to the LB medium. NT102 is an E. coli strain series that harbors pRB102 or its derivatives instead of pRB101. The growth rates of the NT102 variants were determined by measuring the optical density at 600 nm every 15 min using a plate reader (Molecular Devices, Inc.). Construction of the Plasmid Library of 23 S rRNA Randomized at Position H69—G1910-C1920 of H69 in 23 S rRNA (Fig. 1B) encoded in pRB102 was flipped by QuikChange site-directed mutagenesis (Stratagene) according to the manufacturer's instructions using a set of primers C1910-G1920F (5′-cggcggccctaactataagggtcctaaggtagcg-3′) and C1910-G1920R (5′-ccttaggacccttatagttagggccgccgtttaccgggg-3′). This resulted in the construction of pRB102-(C1910-G1920), which was used as a template for PCR randomization to distinguish the selected variants from contamination of the template plasmid after SSER. The resulting plasmid was checked to ensure that the mutations did not confer dominant lethality by introducing it into NT101 (by the process described below). pRB102-(C1910-G1920) was hypermethylated by DNA methyltransferases, M-AluI, M-HaeIII, and M-HapII (Takara BIO, Inc.) as described 4N. S. Sato, N. Hirabayashi, and T. Suzuki, submitted for publication. and employed as a template for PCR randomization to reduce the background for SSER selection. In the first PCR, a set of primers complementary to sequences just outside the target sequence, gap-F (5′-ggtcctaaggtagcgaaattccttgtcggg-3′) and gap-R (5′-ggccgccgtttaccggggcttcgatcaag-3′), was employed to generate gapped pRB102. This enhances the efficiency of PCR randomization and reduces the background for SSER selection. The gapped pRB102-(C1910-G1920) was then gel-purified and used as a template for PCR randomization. The N primer (5′-gctaccttaggaccgtNNNNNNNacggccgccgtttaccgggg-3′) and the gap-F primer were employed to randomize seven bases in the loop of H69. 4N. S. Sato, N. Hirabayashi, and T. Suzuki, submitted for publication. C1910-G1920 mutations were changed back to the original sequence in this step. To obtain enough material for the randomized library, the reaction was performed in 56 tubes (total 2800 μl). After PCR, the reaction mixture was treated with DpnI (New England Biolabs) and λ exonuclease (New England Biolabs) to digest the template plasmid. The products were then cleaned up with QIA-Quik (Qiagen) and checked by agarose gel electrophoresis. Unbiased distribution of four nucleotides at each randomized position was confirmed by direct sequencing of the plasmid library before selection. SSER Selection—Detailed procedure of SSER was described in Sato et al. 4N. S. Sato, N. Hirabayashi, and T. Suzuki, submitted for publication. NT101 was then transformed by the randomized library and spread onto LB plates (56 plates in total) containing Spc/Km. Transformants (896 colonies) were picked to make a suspension in LB broth and then spotted onto LB plates containing Spc/Km/sucrose to force plasmid replacement. When variants showing slow growth phenotypes were picked, their resulting cell suspensions were first spotted and cultivated on LB plates containing Spc/Km (instead of being spotted directly onto LB-sucrose plates) before transfer to LB-sucrose plates. Sucrose-resistant colonies (48 colonies) (NT102 variants) were then cultured in 2× LB broth to make mini-preps of plasmids for sequencing. To check for plasmid replacement, each transformant was spotted onto two LB plates containing Spc/Amp and Spc/Km/sucrose. No growth of the spotted cells on the Amp plate demonstrated complete plasmid replacement. Functional variants were sequenced by the ABI Prism 3100 Genetic Analyzer (Applied Biosystems). B-scan Analysis and Deletion-Insertion Mutations—To analyze the functional importance of the conserved loop of H69 (1912-1918), we replaced these positions with the other three bases by QuikChange site-directed PCR mutagenesis (Stratagene) using a set of mixed primers. Thus, if the targeted position has A, G, C, or T, it was replaced with B (G, C, and T), H (A, C, and T), D (A, G, and T) or V (A, G, and C), respectively. The primer sets employed were B1912F (5′-ccggtaaacggcggccgtBactataacggtcctaagg-3′ and B1912R (5′-ccttaggaccgttatagtVacggccgccgtttaccgg-3′), B1913F (5′-cggtaaacggcggccgtaBctataacggtcctaaggtagc-3′) and B1913R (5′-gctaccttaggaccgttatagVtacggccgccgtttacc-3′), D1914F (5′-ggtaaacggcggccgtaaDtataacggtcctaaggtagc-3′) and D1914R (5′-gctaccttaggaccgttataHttacggccgccgtttacc-3′), V1915F (5′-gtaaacggcggccgtaacVataacggtcctaaggtagc-3′) and V1915R (5′-gctaccttaggaccgttatBgttacggccgccgtttac-3′), B1916F (5′-gtaaacggcggccgtaactBtaacggtcctaaggtagc-3′) and B1916R (5′-gctaccttaggaccgttaVagttacggccgccgtttac-3′), V1917F (5′-gtaaacggcggccgtaactaVaacggtcctaaggtagc-3′) and V1917R (5′-gctaccttaggaccgttBtagttacggccgccgtttac-3′), B1918F (5′-gtaaacggcggccgtaactatBacggtcctaaggtagcg-3′) and B1918R (5′-cgctaccttaggaccgtVatagttacggccgccgttta-3′). The primer sets for deletion and insertion mutations are Δ1913F (5′-cggtaaacggcggccgtactataacggtcctaaggtagc-3′) and Δ1913R (5′-gctaccttaggaccgttatagtacggccgccgtttacc-3′), Δ1916F (5′-gtaaacggcggccgtaacttaacggtcctaaggtagc-3′) and Δ1916R (5′-gctaccttaggaccgttaagttacggccgccgtttac-3′), Δ(1910-1920)F (5′-cggcggcctaactataaggtcctaaggtagcg-3′) and Δ(1910-1920)R (5′-ccttaggaccttatagttaggccgccgtttaccgggg-3′), insN1913-4F (5′-ggtaaacggcggccgtaaNctataacggtcctaaggtagc-3′) and insN1913-4R (5′-gctaccttaggaccgttatagNttacggccgccgtttacc-3′). As with the SSER method, the resulting pRB102-derived library bearing mutations at each position was checked by agarose gel electrophoresis and then introduced into NT101. Functional variants were selected on sucrose plates and sequenced. If the target base was essential for ribosomal function, no variants could be obtained. Subunit Association—NT102 of each variant was cultured in 50 ml of 2× LB medium until 0.5 A600 and harvested. The cultured cells were dissolved in RBS buffer (20 mm Hepes-HCl (pH 7.6), 30 mm NH4Cl, 6 mm Mg(OAc)2, and 6 mm 2-mercaptoethanol) with 0.75 mg/ml lysozyme. After freeze-thawing by liquid N2, 200 μl of the lysate was loaded onto a 10-40% (w/v) sucrose gradient and centrifuged for 14 h at 20,000 rpm in a SW28 rotor. As for Mg2+ titration in Fig. 2B, Mg2+ concentration was adjusted with RBS buffer (6, 10, and 15 mm). Sucrose density gradient (SDG) centrifugation profiles of ribosomal subunits were monitored by using a Bio-mini UV monitor (ATTO, Japan). Translational Fidelity Measured by β-Galactosidase Activity—The mobile plasmid pNT3-lacZ (Ampr) (kindly provided by Dr. Akiko Nishimura) (15Saka K. Tadenuma M. Nakade S. Tanaka N. Sugawara H. Nishikawa K. Ichiyoshi N. Kitagawa M. Mori H. Ogasawara N. Nishimura A. DNA Res. 2005; 12: 63-68Crossref PubMed Scopus (71) Google Scholar) was employed to construct a series of lacZ reporters for measuring the recoding activities of ribosome variants. Windows of test sequences for frameshift or read-though were introduced at the beginning of the lacZ open reading frame in pNT3-lacZ. The resultant plasmids pNT3-lacZ (+1), pNT3-lacZ (-1), pNT3-lacZ (UGA), and pNT3-lacZ (UAG) were used to measure +1 or -1 frameshift and UGA or UAG stop codon read-through, respectively. E. coli JA200 (F+ Δ(trpE)5 recA thr-1 leu-6 lacY thigal xyl ara mtl), which was used as a donor strain for plasmid transfer, was transformed with each reporter plasmid. Each E. coli NT102 bearing a pRB102 with a H69 variation was mated with the donor strain harboring each of the lacZ reporter plasmids transferred through the pilus during conjugation (15Saka K. Tadenuma M. Nakade S. Tanaka N. Sugawara H. Nishikawa K. Ichiyoshi N. Kitagawa M. Mori H. Ogasawara N. Nishimura A. DNA Res. 2005; 12: 63-68Crossref PubMed Scopus (71) Google Scholar). For mating, precultured JA200-pNT-lacZ (10 μl) and NT102 (10 μl) were mixed and diluted in 200 μl of 2× LB without antibiotics. After incubation for 4 h at 37 °C, NT102 harboring the pNT-lacZ reporter was selected on LB plates containing Spc, Km, Amp, and 5% sucrose. Single colonies were grown to confluence, diluted (1:30) into fresh medium, and cultured at 37 °C until logarithmic phase; the cells were harvested at 0.5 A600. The β-galactosidase assay was performed according to Miller (16Miller J.H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. 1992; (Cold Spring Harbor Laboratory Press, Plainview, NY): 72-74Google Scholar). A detailed description for this method will be published elsewhere. Preparation of Ribosomes and Elongation Factors—Ribosomes were prepared from variant strains as described (17Spedding G. Isolation and Analysis of Ribosomes from Prokaryotes, Eukaryotes, and Organelles, Ribosomes and Protein Synthesis, A Practical Approach. 1990; (Oxford University Press, New York): 1-30Google Scholar) with slight modifications. Each E. coli strain grown up to 0.5 A600 was harvested, ground with Al2O3, then dissolved in a buffer consisting of 20 mm Hepes-KOH (pH 7.6), 30 mm NH4Cl, 10 mm Mg(Ac)2,and 6 mm 2-mercaptoethanol. The lysate was subjected to ultracentrifugation to obtain the crude ribosome. 70 S tight-coupled (TC) ribosomes were then purified from the crude ribosome preparation by 14-16 h of ultracentrifugation in a 6-38% (w/v) SDG as described (18Hanada T. Suzuki T. Yokogawa T. Takemoto-Hori C. Sprinzl M. Watanabe K. Genes Cells. 2001; 6: 1019-1030Crossref PubMed Scopus (47) Google Scholar). Recombinant elongation factors were prepared as described (19Shimizu Y. Inoue A. Tomari Y. Suzuki T. Yokogawa T. Nishikawa K. Ueda T. Nat. Biotechnol. 2001; 19: 751-755Crossref PubMed Scopus (1343) Google Scholar). Poly(U)-programmed Polyphenylalanine Synthesis—In vitro translation was performed at 37 °C in 80 μl of reaction mixture consisting of 50 mm Tris-HCl (pH 7.5), 1 mm dithiothreitol, 60 mm KCl, 6.5 mm MgCl2, 0.5 mg/ml poly(U), 0.1 mm spermine, 2.5 mm phosphoenolpyruvic acid, 2.5 unit/ml pyruvate kinase, 0.5 mm GTP, 0.6 μm E. coli EF-Tu, 0.6 μm E. coli EF-G, 0.15 μm E. coli EF-Ts, 0.075 μm 70 S ribosome, and 0.15 μm [14C]Phe-tRNAPhe. 10 μl of the reaction mixture was spotted onto filtration paper (Whatman No. 3MM) every minute. Trichloroacetic acid-insoluble product was quantified by liquid scintillation counting (ALOKA) after Phe-tRNAPhe deacylation by heating at 80 °C for 30 min. In the experiment shown in Fig. 4B, the concentration of [14C]Phe-tRNAphe ranged from 0 to 0.9 μm. Amounts of [14C]Phe incorporated in 10 μl of the reaction mixture at 6 min were plotted. tRNA Binding to A and P Sites—For P-site tRNA binding (Fig. 5A), 12.5 pmol of 70 S ribosome, 2.5 pmol of Ac-[14C]Phe-tRNAPhe, and 2.5 μg of poly(U) in a buffer containing 50 mm Tris-HCl (pH 7.5), 6.5 mm MgCl2, 60 mm KCl, 1 mm dithiothreitol, and 0.5 mm spermine were incubated at 37 °C for 15 min. An aliquot was then spotted onto a nitrocellulose filter (Advantec) and washed with 5 ml of wash buffer (50 mm Tris-HCl (pH 7.5), 6.5 mm MgCl2, 60 mm KCl, 1 mm dithiothreitol, and 0.5 mm spermine). Ac-[14C]Phe-tRNAPhe bound on the P-site was quantified by liquid scintillation counting (ALOKA). Concerning A-site tRNA binding (Fig. 5B), the P-site of the ribosome was occupied by deacyl-tRNAPhe in a mixture (10 μl) consisting of 5 pmol of E. coli 70 S TC ribosome, 2 μg of poly(U), 10 pmol of E. coli tRNAPhe, 50 mm Tris-HCl (pH 7.5), 6.5 mm MgCl2, 60 mm KCl, 1 mm dithiothreitol, and 0.5 mm spermine, which was incubated at 37 °C for 20 min. Then 10 μl of aa-tRNA mixture consisting of 2.5 mm phosphoenolpyruvic acid, 2.5 unit/ml pyruvate kinase, 1 mm GTP, 10 pmol of [14C]Phe-tRNAPhe, and 12.5 pmol of E. coli EF-Tu was added to the ribosome mixture and incubated at 37 °C for 10 min. The following step was as described above. Primer Extension Analysis to Detect Post-transcriptional Modifications in 23 S rRNA—Isolation of total RNA and primer extension to detect pseudouridine (Ψ) were performed basically as described (20Ofengand J. Del Campo M. Kaya Y. Methods. 2001; 25: 365-373Crossref PubMed Scopus (70) Google Scholar). Dried total RNA (4 μg) of each strain was dissolved in 30 μl of CMC solution (0.17 m N-cyclohexyl-N′-β-(4-methylmorpholinium)ethylcarbodiimide p-tosylate (CMC), 7 m urea, 50 mm Bicine, and 4 mm EDTA) and incubated at 37 °C for 20 min. Stop solution (100 μl) consisting of 0.3 m NaOAc (pH 5.6) and 0.1 m EDTA was added to the reaction mixture, then 700 μl of EtOH was added to precipitate the RNA. After rinsing twice with 70% EtOH, the pellet was dissolved in 40 μl of 50 mm Na2CO3 (pH 10.4) at 37 °C for 4 h. After EtOH precipitation, it was dissolved in 10 μl of double-distilled dH2O. 5′-32P-Labeled primer, 5′-aatttcgctaccttaggaccg-3′, complementary to positions 1920-1940 in 23 S rRNA was used for the primer extension. SuperScript III RNase H- reverse transcriptase (Invitrogen) was used for reverse transcription. The cDNAs resolved on 15% polyacrylamide gel containing 7 m urea was detected by fluoroimager (FLA-7000; Fujifilm). Comprehensive Genetic Selection of Functional Sequences of the H69 Loop in 23 S rRNA—To analyze the functional importance of the highly conserved loop sequence of H69 in 23 S rRNA, we employed our new genetic method (SSER).4 This system allows us to rapidly identify functional sequences in the cell among randomized sequences. Seven bases (1912-1918) in the loop of H69 in 23 S rRNA were completely randomized on pRB102 by a PCR-based method to construct a plasmid library with 16,384 sequence variations (Fig. 1B). Then we carried out large scale transformation of E. coli NT101 with each member of the library and selected for kanamycin-resistant transformants. If the incorporated plasmid had a toxic sequence that resulted in a dominant lethal pheno-type, a transformant would not be obtained. Because H69 is a critical site for ribosomal function, most of the sequences in the library must have been excluded during this step. Transformants on the kanamycin plate showed different colony sizes (data not shown), indicating that each cell contains a sequence that results in altered ribosomal activity. The kanamycin-resistant cells contain both pRB101 and pRB102 from the library. To drive plasmid replacement, each cell was picked and spotted onto selection plates containing kanamycin and sucrose. If the incorporated pRB102 plasmid had a functional sequence for ribosomal activity, it rapidly eliminated the pRB101 rescue plasmid, thus yielding sucrose-resistant cells (NT102 derivatives) because these two plasmids share the same replicon and are, thus, incompatible. Besides, if the incorporated plasmid had a non-functional or very weak functional sequence for ribosomal activity, the rescue plasmid could not be replaced by the introduced plasmid and the transformant became sensitive to sucrose due to the sacB gene. About 900 colonies on kanamycin plates were picked and spotted onto sucrose plates. The functional sequences of the H69 loop in pRB102 from the sucrose-resistant cells (NT102) were sequenced. Despite the high conservation of H69-loop sequence, 13 functional variants were obtained (Table 1). A1912 and U1917 were completely conserved in all of the selected variants. This confirms that A1912 and U1917 are essential residues for ribosomal function. AACUAUA (variant 1), which is the original sequence of E. coli 23 S rRNA as well as the conserved sequences in bacterial, archaebacterial, and eukaryotic rRNAs, was actually selected from 16,384 sequence variations. An A1913G variation was found in variants 7 and 12. G1913 is a naturally occurring variation found in Streptomyces galbus and Streptomyces mashuensis. C1914 could be replaced with A or G, although C1914 is completely conserved in bacterial and archaebacterial rRNAs. A1915 and C1915 were selected as functional variations. In fact, A1915 and C1915 are found in rRNAs from Wolbachieae and Aerophrum (Archaea), respectively. A1916 was found to be replaceable by any of three other bases. C1916 is a naturally occurring variation found in Wolbachieae, Staphylococcus piscifermentans, and Crenarchaeota (Archaea). A1918G variation was found in variants 6, 9, and 13. G1918 is a conserved base in eukaryotic rRNAs. In addition, G1918 is a natural variation found in some bacterial and archaebacterial rRNAs. Thus, the consensus sequence of the H69 loop was ARVHNUR (Table 1). 5The nomenclature for mixed bases used in this study obey the rules previously established in the literature (13Cornish-Bowden A. Nucleic Acids Res. 1985; 13: 3021-3030Crossref PubMed Scopus (351) Google Scholar), namely, R is A or G, Y is U or C, B is all bases except A, D is all bases except C, H is all bases except G, V is all bases except U, and N is all bases (A, U, G, or C). TABLE 1Functional sequences (1912-1918) in the H69 loop of E. coli 23 S rRNA as selected by SSER from randomized rRNA libraries Invariant or semiconserved residues selected in this study are shown in red or orange respectively. A1915 is colored blue. RNA modifications are ignored. The nomenclature used for the mixed bases obeys the rules previously established in the literature (13Cornish-Bowden A. Nucleic Acids Res. 1985; 13: 3021-3030Crossref PubMed Scopus (351) Google Scholar). Relative growth rate (RGR) was obtained by calculating the ratio of the doubling time of the mutant to that of the wild type. Consensus sequences of SSER and B-scan analysis are shown in the bottom. Open table in a new tab To confirm this result, we adopted another method to test the essentiality of each base of the H69 loop for ribosomal function. Each nucleotide position was replaced with any of three other bases by PCR mutagenesis using a mixed primer. In other words, if the target position was A, it was" @default.
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• W2023798478 title "Conserved Loop Sequence of Helix 69 in Escherichia coli 23 S rRNA Is Involved in A-site tRNA Binding and Translational Fidelity" @default.
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