Abstract
Several C----U transitions and small deletions were introduced into the conserved region centered on base C1400 in Escherichia coli 16S rRNA by in vitro mutagenesis. The mutations were placed within rrnB operons on multicopy plasmids under the transcriptional regulation of either the normal rrnB P1P2 promoters or the temperature-inducible PL promoter from bacteriophage lambda and introduced into E. coli hosts. When expressed from the P1P2 promoters, several of the mutant 16S rRNAs impaired cell growth while others, including one in which U replaced C at position 1400 within the ribosomal decoding site, had little or no effect on cell doubling time. However, C----U transitions at positions 1395 and 1407, as well as the deletion of C1400, appeared to render their hosts inviable. Cells in which these mutations were expressed from the lambdaPL promoter died within four generations after induction. Unexpectedly, the lethal phenotype was suppressed intragenically by replacement of G1505 with A, C or U. Suppression may alleviate a functional defect in 30S subunits containing the U1395, U1407 or deltaC1400 mutations.
Full text
PDF

















Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bowman C. M., Dahlberg J. E., Ikemura T., Konisky J., Nomura M. Specific inactivation of 16S ribosomal RNA induced by colicin E3 in vivo. Proc Natl Acad Sci U S A. 1971 May;68(5):964–968. doi: 10.1073/pnas.68.5.964. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brimacombe R., Atmadja J., Stiege W., Schüler D. A detailed model of the three-dimensional structure of Escherichia coli 16 S ribosomal RNA in situ in the 30 S subunit. J Mol Biol. 1988 Jan 5;199(1):115–136. doi: 10.1016/0022-2836(88)90383-x. [DOI] [PubMed] [Google Scholar]
- Brosius J., Dull T. J., Sleeter D. D., Noller H. F. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol. 1981 May 15;148(2):107–127. doi: 10.1016/0022-2836(81)90508-8. [DOI] [PubMed] [Google Scholar]
- Cole J. R., Olsson C. L., Hershey J. W., Grunberg-Manago M., Nomura M. Feedback regulation of rRNA synthesis in Escherichia coli. Requirement for initiation factor IF2. J Mol Biol. 1987 Dec 5;198(3):383–392. doi: 10.1016/0022-2836(87)90288-9. [DOI] [PubMed] [Google Scholar]
- Everett R. D., Chambon P. A rapid and efficient method for region- and strand-specific mutagenesis of cloned DNA. EMBO J. 1982;1(4):433–437. doi: 10.1002/j.1460-2075.1982.tb01187.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gourse R. L., Takebe Y., Sharrock R. A., Nomura M. Feedback regulation of rRNA and tRNA synthesis and accumulation of free ribosomes after conditional expression of rRNA genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1069–1073. doi: 10.1073/pnas.82.4.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gourse R. L., de Boer H. A., Nomura M. DNA determinants of rRNA synthesis in E. coli: growth rate dependent regulation, feedback inhibition, upstream activation, antitermination. Cell. 1986 Jan 17;44(1):197–205. doi: 10.1016/0092-8674(86)90498-8. [DOI] [PubMed] [Google Scholar]
- Gregory R. J., Zimmermann R. A. Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16S ribosomal RNA from Escherichia coli. Nucleic Acids Res. 1986 Jul 25;14(14):5761–5776. doi: 10.1093/nar/14.14.5761. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jemiolo D. K., Zwieb C., Dahlberg A. E. Point mutations in the 3' minor domain of 16S rRNA of E.coli. Nucleic Acids Res. 1985 Dec 9;13(23):8631–8643. doi: 10.1093/nar/13.23.8631. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jinks-Robertson S., Gourse R. L., Nomura M. Expression of rRNA and tRNA genes in Escherichia coli: evidence for feedback regulation by products of rRNA operons. Cell. 1983 Jul;33(3):865–876. doi: 10.1016/0092-8674(83)90029-6. [DOI] [PubMed] [Google Scholar]
- Kalderon D., Oostra B. A., Ely B. K., Smith A. E. Deletion loop mutagenesis: a novel method for the construction of point mutations using deletion mutants. Nucleic Acids Res. 1982 Sep 11;10(17):5161–5171. doi: 10.1093/nar/10.17.5161. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Krzyzosiak W., Denman R., Nurse K., Hellmann W., Boublik M., Gehrke C. W., Agris P. F., Ofengand J. In vitro synthesis of 16S ribosomal RNA containing single base changes and assembly into a functional 30S ribosome. Biochemistry. 1987 Apr 21;26(8):2353–2364. doi: 10.1021/bi00382a042. [DOI] [PubMed] [Google Scholar]
- Meier N., Göringer H. U., Kleuvers B., Scheibe U., Eberle J., Szymkowiak C., Zacharias M., Wagner R. The importance of individual nucleotides for the structure and function of rRNA molecules in E. coli. A mutagenesis study. FEBS Lett. 1986 Aug 11;204(1):89–95. doi: 10.1016/0014-5793(86)81392-8. [DOI] [PubMed] [Google Scholar]
- Meier N., Wagner R. Binding of tRNA alters the chemical accessibility of nucleotides within the large ribosomal RNAs of E. coli ribosomes. Nucleic Acids Res. 1984 Feb 10;12(3):1473–1487. doi: 10.1093/nar/12.3.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
- Moazed D., Noller H. F. Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes. Cell. 1986 Dec 26;47(6):985–994. doi: 10.1016/0092-8674(86)90813-5. [DOI] [PubMed] [Google Scholar]
- Moazed D., Van Stolk B. J., Douthwaite S., Noller H. F. Interconversion of active and inactive 30 S ribosomal subunits is accompanied by a conformational change in the decoding region of 16 S rRNA. J Mol Biol. 1986 Oct 5;191(3):483–493. doi: 10.1016/0022-2836(86)90143-9. [DOI] [PubMed] [Google Scholar]
- Noller H. F. Structure of ribosomal RNA. Annu Rev Biochem. 1984;53:119–162. doi: 10.1146/annurev.bi.53.070184.001003. [DOI] [PubMed] [Google Scholar]
- Prince J. B., Taylor B. H., Thurlow D. L., Ofengand J., Zimmermann R. A. Covalent crosslinking of tRNA1Val to 16S RNA at the ribosomal P site: identification of crosslinked residues. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5450–5454. doi: 10.1073/pnas.79.18.5450. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wolters J., Erdmann V. A. Compilation of 5S rRNA and 5S rRNA gene sequences. Nucleic Acids Res. 1988;16 (Suppl):r1–70. doi: 10.1093/nar/16.suppl.r1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamagishi M., de Boer H. A., Nomura M. Feedback regulation of rRNA synthesis. A mutational alteration in the anti-Shine-Dalgarno region of the 16 S rRNA gene abolishes regulation. J Mol Biol. 1987 Dec 5;198(3):547–550. doi: 10.1016/0022-2836(87)90299-3. [DOI] [PubMed] [Google Scholar]
- Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]