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. 1991 Aug 11;19(15):4259–4265. doi: 10.1093/nar/19.15.4259

Mutations in 16S rRNA in Escherichia coli at methyl-modified sites: G966, C967, and G1207.

D K Jemiolo 1, J S Taurence 1, S Giese 1
PMCID: PMC328571  PMID: 1714565

Abstract

Mutations were constructed at three sites in 16S rRNA in E. coli by oligonucleotide-directed mutagenesis, and cloned into the rrnB operon on either pKK3535 or pNO2680. The mutated bases, G966, C967, and G1207, are located in the 3' major domain of 16S rRNA and are sites post-transcriptionally modified by methylation. We constructed a deletion mutation at C967 (delta 967) and three substitution mutations at each of the following sites: G966, C967, and G1207. By maxicell analysis, we found that all of the mutations were processed normally and incorporated into 30S subunits and 70S ribosomes. We found that delta 967 was a dominant lethal mutation while the substitution mutations at G966 and C967 had no effects on cell growth rate. The mutants C1207 and U1207 were shown to have dominant lethal phenotypes while A1207 had no effect on cell growth rate. These results help to establish the importance of methyl-modified regions to ribosome function.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Brosius J., Ullrich A., Raker M. A., Gray A., Dull T. J., Gutell R. R., Noller H. F. Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli. Plasmid. 1981 Jul;6(1):112–118. doi: 10.1016/0147-619x(81)90058-5. [DOI] [PubMed] [Google Scholar]
  3. Dahlberg A. E. The functional role of ribosomal RNA in protein synthesis. Cell. 1989 May 19;57(4):525–529. doi: 10.1016/0092-8674(89)90122-0. [DOI] [PubMed] [Google Scholar]
  4. Gourse R. L., Stark M. J., Dahlberg A. E. Site-directed mutagenesis of ribosomal RNA. Construction and characterization of deletion mutants. J Mol Biol. 1982 Aug 15;159(3):397–416. doi: 10.1016/0022-2836(82)90291-1. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Jemiolo D. K., Steen R., Stark M. J., Dahlberg A. E. Analysis of plasmid-coded ribosomal RNA maxicell techniques. Methods Enzymol. 1988;164:691–706. doi: 10.1016/s0076-6879(88)64078-x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  11. Leclerc D., Brakier-Gingras L. Study of the function of Escherichia coli ribosomal RNA through site-directed mutagenesis. Biochem Cell Biol. 1990 Jan;68(1):169–179. doi: 10.1139/o90-023. [DOI] [PubMed] [Google Scholar]
  12. Melançon P., Gravel M., Boileau G., Brakier-Gingras L. Reassembly of active 30S ribosomal subunits with an unmethylated in vitro transcribed 16S rRNA. Biochem Cell Biol. 1987 Dec;65(12):1022–1030. doi: 10.1139/o87-134. [DOI] [PubMed] [Google Scholar]
  13. Moazed D., Stern S., Noller H. F. Rapid chemical probing of conformation in 16 S ribosomal RNA and 30 S ribosomal subunits using primer extension. J Mol Biol. 1986 Feb 5;187(3):399–416. doi: 10.1016/0022-2836(86)90441-9. [DOI] [PubMed] [Google Scholar]
  14. Murgola E. J., Hijazi K. A., Göringer H. U., Dahlberg A. E. Mutant 16S ribosomal RNA: a codon-specific translational suppressor. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4162–4165. doi: 10.1073/pnas.85.12.4162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Remaut E., Stanssens P., Fiers W. Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda. Gene. 1981 Oct;15(1):81–93. doi: 10.1016/0378-1119(81)90106-2. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Siehnel R. J., Morgan E. A. Unbalanced rRNA gene dosage and its effects on rRNA and ribosomal-protein synthesis. J Bacteriol. 1985 Aug;163(2):476–486. doi: 10.1128/jb.163.2.476-486.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sigmund C. D., Ettayebi M., Borden A., Morgan E. A. Antibiotic resistance mutations in ribosomal RNA genes of Escherichia coli. Methods Enzymol. 1988;164:673–690. doi: 10.1016/s0076-6879(88)64077-8. [DOI] [PubMed] [Google Scholar]
  19. Stark M. J., Gourse R. L., Dahlberg A. E. Site-directed mutagenesis of ribosomal RNA. Analysis of ribosomal RNA deletion mutants using maxicells. J Mol Biol. 1982 Aug 15;159(3):417–439. doi: 10.1016/0022-2836(82)90292-3. [DOI] [PubMed] [Google Scholar]
  20. Thomas C. L., Gregory R. J., Winslow G., Muto A., Zimmermann R. A. Mutations within the decoding site of Escherichia coli 16S rRNA: growth rate impairment, lethality and intragenic suppression. Nucleic Acids Res. 1988 Aug 25;16(16):8129–8146. doi: 10.1093/nar/16.16.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Woese C. R., Gutell R., Gupta R., Noller H. F. Detailed analysis of the higher-order structure of 16S-like ribosomal ribonucleic acids. Microbiol Rev. 1983 Dec;47(4):621–669. doi: 10.1128/mr.47.4.621-669.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

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