Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Sep;173(18):5831–5836. doi: 10.1128/jb.173.18.5831-5836.1991

Mutations that affect activity of the Rhizobium meliloti trpE(G) promoter in Rhizobium meliloti and Escherichia coli.

Y M Bae 1, G V Stauffer 1
PMCID: PMC208316  PMID: 1885552

Abstract

The cloned Rhizobium meliloti trpE(G) gene is not expressed in Escherichia coli. Oligonucleotide-directed mutagenesis was used to introduce base substitution mutations in the promoter region of this gene. Three separate mutations that increased homology of the putative -10 region of this promoter with the E. coli -10 promoter consensus sequence by 1 bp converted this promoter to an active promoter in E. coli. A deletion extending to position -43 from the 5' side had a minor effect on transcription in R. meliloti. However, transcription was nearly eliminated when a deletion extended to position -33, indicating that the crucial domain of the R. meliloti trpE(G) promoter begins in the region downstream of position -43. The R. meliloti trpE(G) promoter has two regions that show homology with the E. coli -35 and -10 promoter consensus sequences. Mutations in these putative -35 and -10 regions, but not in the spacer region, affected promoter strength in R. meliloti. By comparing four known R. meliloti promoter sequences, we identified a highly conserved trimer near position -35 (5'-TTG-3') but no noticeably conserved sequence near position -10.

Full text

PDF
5836

Selected References

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

  1. Bae Y. M., Crawford I. P. The Rhizobium meliloti trpE(G) gene is regulated by attenuation, and its product, anthranilate synthase, is regulated by feedback inhibition. J Bacteriol. 1990 Jun;172(6):3318–3327. doi: 10.1128/jb.172.6.3318-3327.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bae Y. M., Holmgren E., Crawford I. P. Rhizobium meliloti anthranilate synthase gene: cloning, sequence, and expression in Escherichia coli. J Bacteriol. 1989 Jun;171(6):3471–3478. doi: 10.1128/jb.171.6.3471-3478.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bae Y. M., Stauffer G. V. Genetic analysis of the attenuator of the Rhizobium meliloti trpE(G) gene. J Bacteriol. 1991 Jun;173(11):3382–3388. doi: 10.1128/jb.173.11.3382-3388.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Better M., Helinski D. R. Isolation and characterization of the recA gene of Rhizobium meliloti. J Bacteriol. 1983 Jul;155(1):311–316. doi: 10.1128/jb.155.1.311-316.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boesten B., Lenzen G., Danchin A., O'Gara F. Nucleotide sequence of a tRNA(leu)CAG gene from Rhizobium meliloti. Gene. 1987;55(1):153–156. doi: 10.1016/0378-1119(87)90259-9. [DOI] [PubMed] [Google Scholar]
  6. Bossi L., Smith D. M. Conformational change in the DNA associated with an unusual promoter mutation in a tRNA operon of Salmonella. Cell. 1984 Dec;39(3 Pt 2):643–652. doi: 10.1016/0092-8674(84)90471-9. [DOI] [PubMed] [Google Scholar]
  7. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chung C. T., Niemela S. L., Miller R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2172–2175. doi: 10.1073/pnas.86.7.2172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fisher R. F., Brierley H. L., Mulligan J. T., Long S. R. Transcription of Rhizobium meliloti nodulation genes. Identification of a nodD transcription initiation site in vitro and in vivo. J Biol Chem. 1987 May 15;262(14):6849–6855. [PubMed] [Google Scholar]
  10. Fisher R. F., Long S. R. DNA footprint analysis of the transcriptional activator proteins NodD1 and NodD3 on inducible nod gene promoters. J Bacteriol. 1989 Oct;171(10):5492–5502. doi: 10.1128/jb.171.10.5492-5502.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Graña D., Gardella T., Susskind M. M. The effects of mutations in the ant promoter of phage P22 depend on context. Genetics. 1988 Oct;120(2):319–327. doi: 10.1093/genetics/120.2.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harley C. B., Reynolds R. P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. doi: 10.1093/nar/15.5.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Helmann J. D., Chamberlin M. J. DNA sequence analysis suggests that expression of flagellar and chemotaxis genes in Escherichia coli and Salmonella typhimurium is controlled by an alternative sigma factor. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6422–6424. doi: 10.1073/pnas.84.18.6422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Inouye S., Inouye M. Up-promoter mutations in the lpp gene of Escherichia coli. Nucleic Acids Res. 1985 May 10;13(9):3101–3110. doi: 10.1093/nar/13.9.3101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jacquet M. A., Reiss C. Transcription in vivo directed by consensus sequences of E.coli promoters: their context heavily affects efficiencies and start sites. Nucleic Acids Res. 1990 Mar 11;18(5):1137–1143. doi: 10.1093/nar/18.5.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Johnston A. W., Bibb M. J., Beringer J. E. Tryptophan genes in Rhizobium--their organization and their transfer to other bacterial genera. Mol Gen Genet. 1978 Oct 24;165(3):323–330. doi: 10.1007/BF00332533. [DOI] [PubMed] [Google Scholar]
  18. Koo H. S., Wu H. M., Crothers D. M. DNA bending at adenine . thymine tracts. Nature. 1986 Apr 10;320(6062):501–506. doi: 10.1038/320501a0. [DOI] [PubMed] [Google Scholar]
  19. Leong S. A., Williams P. H., Ditta G. S. Analysis of the 5' regulatory region of the gene for delta-aminolevulinic acid synthetase of Rhizobium meliloti. Nucleic Acids Res. 1985 Aug 26;13(16):5965–5976. doi: 10.1093/nar/13.16.5965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Long S. R. Rhizobium genetics. Annu Rev Genet. 1989;23:483–506. doi: 10.1146/annurev.ge.23.120189.002411. [DOI] [PubMed] [Google Scholar]
  21. Mandecki W., Goldman R. A., Powell B. S., Caruthers M. H. lac Up-promoter mutants with increased homology to the consensus promoter sequence. J Bacteriol. 1985 Dec;164(3):1353–1355. doi: 10.1128/jb.164.3.1353-1355.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McClure W. R. Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem. 1985;54:171–204. doi: 10.1146/annurev.bi.54.070185.001131. [DOI] [PubMed] [Google Scholar]
  23. Nielsen B. L., Brown L. R. Purification and subunit characterization of Rhizobium meliloti RNA polymerase. J Bacteriol. 1985 May;162(2):645–650. doi: 10.1128/jb.162.2.645-650.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pleier E., Schmitt R. Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti. J Bacteriol. 1989 Mar;171(3):1467–1475. doi: 10.1128/jb.171.3.1467-1475.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Su W., Porter S., Kustu S., Echols H. DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial glnA promoter. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5504–5508. doi: 10.1073/pnas.87.14.5504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Szoke P. A., Allen T. L., deHaseth P. L. Promoter recognition by Escherichia coli RNA polymerase: effects of base substitutions in the -10 and -35 regions. Biochemistry. 1987 Sep 22;26(19):6188–6194. doi: 10.1021/bi00393a035. [DOI] [PubMed] [Google Scholar]
  28. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  29. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  30. von Hippel P. H., Bear D. G., Morgan W. D., McSwiggen J. A. Protein-nucleic acid interactions in transcription: a molecular analysis. Annu Rev Biochem. 1984;53:389–446. doi: 10.1146/annurev.bi.53.070184.002133. [DOI] [PubMed] [Google Scholar]

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

RESOURCES