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. 1987 Jul;84(14):4757–4761. doi: 10.1073/pnas.84.14.4757

A single base change in the Shine-Dalgarno region of 16S rRNA of Escherichia coli affects translation of many proteins.

W F Jacob, M Santer, A E Dahlberg
PMCID: PMC305184  PMID: 2440027

Abstract

A single base mutation was constructed at position 1538 of Escherichia coli 16S rRNA, changing a cytidine to a uridine. This position is in the Shine-Dalgarno region, thought to be involved in base-pairing to mRNA during initiation of protein synthesis. The mutation was constructed by using a synthetic oligodeoxynucleotide that differs in sequence by one base from the wild-type sequence of 16S rRNA. This oligonucleotide was used as a primer on single-stranded DNA of phage M13, into which was cloned a specific region of DNA encoding 16S rRNA. The mutation is lethal when expressed from the normal promoters of rRNA operons, P1 and P2, in a high-copy-number plasmid. Expression can be repressed by a temperature-sensitive repressor, cI857, in combination with the bacteriophage lambda PL promoter. Induction of transcription by temperature shift yields mutant 16S rRNA that is processed and assembled into functional ribosomal subunits. The presence of mutant ribosomes retards cell growth and dramatically alters incorporation of [35S]methionine into a large proportion of the cellular proteins. The change in level of synthesis of individual proteins correlates with the change in base-pairing between mutant rRNA and the Shine-Dalgarno region of the mRNA.

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Selected References

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

  1. Barker D. G., Ebel J. P., Jakes R., Bruton C. J. Methionyl-tRNA synthetase from Escherichia coli. Primary structure of the active crystallised tryptic fragment. Eur J Biochem. 1982 Oct;127(3):449–457. [PubMed] [Google Scholar]
  2. Bolivar F., Backman K. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 1979;68:245–267. doi: 10.1016/0076-6879(79)68018-7. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Chapman N. M., Noller H. F. Protection of specific sites in 16 S RNA from chemical modification by association of 30 S and 50 S ribosomes. J Mol Biol. 1977 Jan 5;109(1):131–149. doi: 10.1016/s0022-2836(77)80049-1. [DOI] [PubMed] [Google Scholar]
  6. Dahlberg A. E., Dahlberg J. E. Binding of ribosomal protein S1 of Escherichia coli to the 3' end of 16S rRNA. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2940–2944. doi: 10.1073/pnas.72.8.2940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dahlberg A. E., Dingman C. W., Peacock A. C. Electrophoretic characterization of bacterial polyribosomes in agarose-acrylamide composite gels. J Mol Biol. 1969 Apr 14;41(1):139–147. doi: 10.1016/0022-2836(69)90131-4. [DOI] [PubMed] [Google Scholar]
  8. Dunn J. J., Buzash-Pollert E., Studier F. W. Mutations of bacteriophage T7 that affect initiation of synthesis of the gene 0.3 protein. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2741–2745. doi: 10.1073/pnas.75.6.2741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gillam S., Astell C. R., Smith M. Site-specific mutagenesis using oligodeoxyribonucleotides: isolation of a phenotypically silent phi X174 mutant, with a specific nucleotide deletion, at very high efficiency. Gene. 1980 Dec;12(1-2):129–137. doi: 10.1016/0378-1119(80)90023-2. [DOI] [PubMed] [Google Scholar]
  10. Gold L., Pribnow D., Schneider T., Shinedling S., Singer B. S., Stormo G. Translational initiation in prokaryotes. Annu Rev Microbiol. 1981;35:365–403. doi: 10.1146/annurev.mi.35.100181.002053. [DOI] [PubMed] [Google Scholar]
  11. Gourse R. L., Stark M. J., Dahlberg A. E. Regions of DNA involved in the stringent control of plasmid-encoded rRNA in vivo. Cell. 1983 Apr;32(4):1347–1354. doi: 10.1016/0092-8674(83)90315-x. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Gren E. J. Recognition of messenger RNA during translational initiation in Escherichia coli. Biochimie. 1984 Jan;66(1):1–29. doi: 10.1016/0300-9084(84)90188-3. [DOI] [PubMed] [Google Scholar]
  15. Hall M. N., Gabay J., Débarbouillé M., Schwartz M. A role for mRNA secondary structure in the control of translation initiation. Nature. 1982 Feb 18;295(5850):616–618. doi: 10.1038/295616a0. [DOI] [PubMed] [Google Scholar]
  16. Hui A., de Boer H. A. Specialized ribosome system: preferential translation of a single mRNA species by a subpopulation of mutated ribosomes in Escherichia coli. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4762–4766. doi: 10.1073/pnas.84.14.4762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kastelein R. A., Berkhout B., Overbeek G. P., van Duin J. Effect of the sequences upstream from the ribosome-binding site on the yield of protein from the cloned gene for phage MS2 coat protein. Gene. 1983 Sep;23(3):245–254. doi: 10.1016/0378-1119(83)90015-x. [DOI] [PubMed] [Google Scholar]
  18. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Markham G. D., DeParasis J., Gatmaitan J. The sequence of metK, the structural gene for S-adenosylmethionine synthetase in Escherichia coli. J Biol Chem. 1984 Dec 10;259(23):14505–14507. [PubMed] [Google Scholar]
  21. 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]
  22. Neidhardt F. C., VanBogelen R. A., Vaughn V. The genetics and regulation of heat-shock proteins. Annu Rev Genet. 1984;18:295–329. doi: 10.1146/annurev.ge.18.120184.001455. [DOI] [PubMed] [Google Scholar]
  23. Neidhardt F. C., Vaughn V., Phillips T. A., Bloch P. L. Gene-protein index of Escherichia coli K-12. Microbiol Rev. 1983 Jun;47(2):231–284. doi: 10.1128/mr.47.2.231-284.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  25. Poldermans B., Bakker H., Van Knippenberg P. H. Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16S ribosomal RNA of Escherichia coli. IV. The effect of the methylgroups on ribosomal subunit interaction. Nucleic Acids Res. 1980 Jan 11;8(1):143–151. doi: 10.1093/nar/8.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Post L. E., Strycharz G. D., Nomura M., Lewis H., Dennis P. P. Nucleotide sequence of the ribosomal protein gene cluster adjacent to the gene for RNA polymerase subunit beta in Escherichia coli. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1697–1701. doi: 10.1073/pnas.76.4.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Scherer G. F., Walkinshaw M. D., Arnott S., Morré D. J. The ribosome binding sites recognized by E. coli ribosomes have regions with signal character in both the leader and protein coding segments. Nucleic Acids Res. 1980 Sep 11;8(17):3895–3907. doi: 10.1093/nar/8.17.3895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schnier J., Kimura M., Foulaki K., Subramanian A. R., Isono K., Wittmann-Liebold B. Primary structure of Escherichia coli ribosomal protein S1 and of its gene rpsA. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1008–1011. doi: 10.1073/pnas.79.4.1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Singer B. S., Gold L., Shinedling S. T., Colkitt M., Hunter L. R., Pribnow D., Nelson M. A. Analysis in vivo of translational mutants of the rIIB cistron of bacteriophage T4. J Mol Biol. 1981 Jul 5;149(3):405–432. doi: 10.1016/0022-2836(81)90479-4. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Steitz J. A., Jakes K. How ribosomes select initiator regions in mRNA: base pair formation between the 3' terminus of 16S rRNA and the mRNA during initiation of protein synthesis in Escherichia coli. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4734–4738. doi: 10.1073/pnas.72.12.4734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stormo G. D., Schneider T. D., Gold L. M. Characterization of translational initiation sites in E. coli. Nucleic Acids Res. 1982 May 11;10(9):2971–2996. doi: 10.1093/nar/10.9.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Taniguchi T., Weissmann C. Inhibition of Qbeta RNA 70S ribosome initiation complex formation by an oligonucleotide complementary to the 3' terminal region of E. coli 16S ribosomal RNA. Nature. 1978 Oct 26;275(5682):770–772. doi: 10.1038/275770a0. [DOI] [PubMed] [Google Scholar]
  36. Van Duin J., Overbeek G. P., Backendorf C. Functional recognition of phage RNA by 30-S ribosomal subunits in the absence of initiator tRNA. Eur J Biochem. 1980 Sep;110(2):593–597. doi: 10.1111/j.1432-1033.1980.tb04903.x. [DOI] [PubMed] [Google Scholar]
  37. Walker J. E., Saraste M., Gay N. J. The unc operon. Nucleotide sequence, regulation and structure of ATP-synthase. Biochim Biophys Acta. 1984 Sep 6;768(2):164–200. doi: 10.1016/0304-4173(84)90003-x. [DOI] [PubMed] [Google Scholar]
  38. Zengel J. M., Archer R. H., Lindahl L. The nucleotide sequence of the Escherichia coli fus gene, coding for elongation factor G. Nucleic Acids Res. 1984 Feb 24;12(4):2181–2192. doi: 10.1093/nar/12.4.2181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. de Boer H. A., Hui A., Comstock L. J., Wong E., Vasser M. Portable Shine-Dalgarno regions: a system for a systematic study of defined alterations of nucleotide sequences within E. coli ribosome binding sites. DNA. 1983;2(3):231–235. doi: 10.1089/dna.1983.2.231. [DOI] [PubMed] [Google Scholar]
  41. van Buul C. P., Visser W., van Knippenberg P. H. Increased translational fidelity caused by the antibiotic kasugamycin and ribosomal ambiguity in mutants harbouring the ksgA gene. FEBS Lett. 1984 Nov 5;177(1):119–124. doi: 10.1016/0014-5793(84)80994-1. [DOI] [PubMed] [Google Scholar]

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