Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Mar;171(3):1485–1491. doi: 10.1128/jb.171.3.1485-1491.1989

Genetic suppression of a dnaG mutation in Escherichia coli.

T Katayama 1, Y Murakami 1, C Wada 1, H Ohmori 1, T Yura 1, T Nagata 1
PMCID: PMC209770  PMID: 2646283

Abstract

Escherichia coli strains with a temperature-sensitive mutation, dnaG2903, in the primase-encoding gene spontaneously reverted to the temperature-insensitive phenotype at a high frequency. Many of the reversions were caused by extragenic sdg suppressors. About 100 independently isolated sdg suppressors were analyzed. They fall into two classes. The sdgA mutations were genetically mapped very close to and upstream of the dnaG gene and were found to be cis dominant. DNA sequencing of two of them revealed that G----A and C----A base substitutions had occurred 43 and 62 bases, respectively, upstream of the dnaG start codon. This region represents a transcriptional terminator thought to contribute to control of dnaG gene expression. The other class of suppressor, sdgB, seemed to comprise mutant alleles in the rpoB gene coding for the beta subunit of RNA polymerase core enzyme. Some of them were initially isolated as rifampin-resistant mutants. Both the sdgA and sdgB suppressors were found to increase the transcriptional activity of dnaG. This finding and other observations led to the proposition that sdgA and sdgB suppress the phenotype caused by dnaG2903 by overproducing the mutated primase; the quantitative oversupply may compensate for the qualitative defect of the dnaG2903 primase. An alternative mechanism of suppression by sdgB is discussed.

Full text

PDF
1489

Images in this article

1489Image
on p.1489

Selected References

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

  1. Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
  2. Atlung T. Allele-specific suppression of dnaA(Ts) mutations by rpoB mutations in Escherichia coli. Mol Gen Genet. 1984;197(1):125–128. doi: 10.1007/BF00327932. [DOI] [PubMed] [Google Scholar]
  3. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev. 1983 Jun;47(2):180–230. doi: 10.1128/mr.47.2.180-230.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baker T. A., Kornberg A. Transcriptional activation of initiation of replication from the E. coli chromosomal origin: an RNA-DNA hybrid near oriC. Cell. 1988 Oct 7;55(1):113–123. doi: 10.1016/0092-8674(88)90014-1. [DOI] [PubMed] [Google Scholar]
  5. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  6. Burton Z. F., Gross C. A., Watanabe K. K., Burgess R. R. The operon that encodes the sigma subunit of RNA polymerase also encodes ribosomal protein S21 and DNA primase in E. coli K12. Cell. 1983 Feb;32(2):335–349. doi: 10.1016/0092-8674(83)90453-1. [DOI] [PubMed] [Google Scholar]
  7. Carl P. L. Escherichia coli mutants with temperature-sensitive synthesis of DNA. Mol Gen Genet. 1970;109(2):107–122. doi: 10.1007/BF00269647. [DOI] [PubMed] [Google Scholar]
  8. Dooley T. P., Tamm J., Polisky B. Isolation and characterization of mutants affecting functional domains of ColE1 RNAI. J Mol Biol. 1985 Nov 5;186(1):87–96. doi: 10.1016/0022-2836(85)90259-1. [DOI] [PubMed] [Google Scholar]
  9. Green M. R., Maniatis T., Melton D. A. Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell. 1983 Mar;32(3):681–694. doi: 10.1016/0092-8674(83)90054-5. [DOI] [PubMed] [Google Scholar]
  10. Gross J. D. DNA replication in bacteria. Curr Top Microbiol Immunol. 1972;57:39–74. doi: 10.1007/978-3-642-65297-4_2. [DOI] [PubMed] [Google Scholar]
  11. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  12. Hirano M., Shigesada K., Imai M. Construction and characterization of plasmid and lambda phage vector systems for study of transcriptional control in Escherichia coli. Gene. 1987;57(1):89–99. doi: 10.1016/0378-1119(87)90180-6. [DOI] [PubMed] [Google Scholar]
  13. Ikeda H., Tomizawa J. I. Transducing fragments in generalized transduction by phage P1. I. Molecular origin of the fragments. J Mol Biol. 1965 Nov;14(1):85–109. doi: 10.1016/s0022-2836(65)80232-7. [DOI] [PubMed] [Google Scholar]
  14. Ikeuchi T., Yura T., Yamagishi H. Genetic and physical studies of lambda transducing bacteriophage carrying the beta subunit gene of the Escherichia coli ribonucleic acid polymerase. J Bacteriol. 1975 Jun;122(3):1247–1256. doi: 10.1128/jb.122.3.1247-1256.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jin D. J., Walter W. A., Gross C. A. Characterization of the termination phenotypes of rifampicin-resistant mutants. J Mol Biol. 1988 Jul 20;202(2):245–253. doi: 10.1016/0022-2836(88)90455-x. [DOI] [PubMed] [Google Scholar]
  16. Kleckner N., Barker D. F., Ross D. G., Botstein D. Properties of the translocatable tetracycline-resistance element Tn10 in Escherichia coli and bacteriophage lambda. Genetics. 1978 Nov;90(3):427–461. doi: 10.1093/genetics/90.3.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lupski J. R., Ruiz A. A., Godson G. N. Promotion, termination, and anti-termination in the rpsU-dnaG-rpoD macromolecular synthesis operon of E. coli K-12. Mol Gen Genet. 1984;195(3):391–401. doi: 10.1007/BF00341439. [DOI] [PubMed] [Google Scholar]
  18. Lupski J. R., Smiley B. L., Blattner F. R., Godson G. N. Cloning and characterization of the Escherichia coli chromosomal region surrounding the dnaG Gene, with a correlated physical and genetic map of dnaG generated via transposon Tn5 mutagenesis. Mol Gen Genet. 1982;185(1):120–128. doi: 10.1007/BF00333800. [DOI] [PubMed] [Google Scholar]
  19. Lupski J. R., Smiley B. L., Godson G. N. Regulation of the rpsU-dnaG-rpoD macromolecular synthesis operon and the initiation of DNA replication in Escherichia coli K-12. Mol Gen Genet. 1983;189(1):48–57. doi: 10.1007/BF00326054. [DOI] [PubMed] [Google Scholar]
  20. Maki S., Kuribayashi M., Miki T., Horiuchi T. An amber replication mutant of F plasmid mapped in the minimal replication region. Mol Gen Genet. 1983;191(2):231–237. doi: 10.1007/BF00334819. [DOI] [PubMed] [Google Scholar]
  21. Marinus M. G., Adelberg E. A. Vegetative Replication and Transfer Replication of Deoxyribonucleic Acid in Temperature-Sensitive Mutants of Escherichia coli K-12. J Bacteriol. 1970 Dec;104(3):1266–1272. doi: 10.1128/jb.104.3.1266-1272.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Murakami Y., Nagata T., Schwarz W., Wada C., Yura T. Novel dnaG mutation in a dnaP mutant of Escherichia coli. J Bacteriol. 1985 May;162(2):830–832. doi: 10.1128/jb.162.2.830-832.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Murakami Y., Ohmori H., Yura T., Nagata T. Requirement of the Escherichia coli dnaA gene function for ori-2-dependent mini-F plasmid replication. J Bacteriol. 1987 Apr;169(4):1724–1730. doi: 10.1128/jb.169.4.1724-1730.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nakamura Y. Amber dnaG mutation exerting a polar effect on the synthesis of RNA polymerase sigma factor in Escherichia coli. Mol Gen Genet. 1984;196(1):179–182. doi: 10.1007/BF00334114. [DOI] [PubMed] [Google Scholar]
  27. Nakamura Y. Hybrid plasmid carrying Escherichia coli genes for the primase (dnaG) and RNA polymerase sigma factor (rpoD); gene organization and control of their expression. Mol Gen Genet. 1980;178(3):487–497. doi: 10.1007/BF00337853. [DOI] [PubMed] [Google Scholar]
  28. Nesin M., Lupski J. R., Svec P., Godson G. N. Possible new genes as revealed by molecular analysis of a 5-kb Escherichia coli chromosomal region 5' to the rpsU-dnaG-rpoD macromolecular-synthesis operon. Gene. 1987;51(2-3):149–161. doi: 10.1016/0378-1119(87)90303-9. [DOI] [PubMed] [Google Scholar]
  29. Norris V., Alliotte T., Jaffé A., D'Ari R. DNA replication termination in Escherichia coli parB (a dnaG allele), parA, and gyrB mutants affected in DNA distribution. J Bacteriol. 1986 Nov;168(2):494–504. doi: 10.1128/jb.168.2.494-504.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Smiley B. L., Lupski J. R., Svec P. S., McMacken R., Godson G. N. Sequences of the Escherichia coli dnaG primase gene and regulation of its expression. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4550–4554. doi: 10.1073/pnas.79.15.4550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wechsler J. A., Gross J. D. Escherichia coli mutants temperature-sensitive for DNA synthesis. Mol Gen Genet. 1971;113(3):273–284. doi: 10.1007/BF00339547. [DOI] [PubMed] [Google Scholar]
  33. Wold M. S., McMacken R. Regulation of expression of the Escherichia coli dnaG gene and amplification of the dnaG primase. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4907–4911. doi: 10.1073/pnas.79.16.4907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yura T., Ishihama A. Genetics of bacterial RNA polymerases. Annu Rev Genet. 1979;13:59–97. doi: 10.1146/annurev.ge.13.120179.000423. [DOI] [PubMed] [Google Scholar]
  35. de Bruijn F. J., Ausubel F. M. The cloning and transposon Tn5 mutagenesis of the glnA region of Klebsiella pneumoniae: identification of glnR, a gene involved in the regulation of the nif and hut operons. Mol Gen Genet. 1981;183(2):289–297. doi: 10.1007/BF00270631. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES