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. 1991 Dec;10(13):4137–4144. doi: 10.1002/j.1460-2075.1991.tb04991.x

Mutations in TrpI binding site II that differentially affect activation of the trpBA promoter of Pseudomonas aeruginosa.

J Gao 1, G N Gussin 1
PMCID: PMC453164  PMID: 1756720

Abstract

In vitro, Pseudomonas aeruginosa TrpI protein activates transcription initiation at the trpBA promoter (trpPB) and represses initiation at its own promoter (trpPI), which diverges from, and overlaps, trpPB. Indoleglycerol phosphate (InGP) reduces the TrpI concentration required for binding to its strong binding site (site I), as measured by repression of trpPI; it also facilitates activation of trpPB, presumably because it enables TrpI to bind to a weaker binding site (site II) and thereby interact with RNA polymerase. The role of site II and InGP in regulation of the two promoters was investigated by constructing site II mutants. A 2 bp substitution affected the ability of TrpI to activate trpPB, but did not significantly affect TrpI binding to site II. A more extensive (8 bp) substitution inhibited TrpI-mediated activation of trpPB and TrpI-mediated protection of site II in a DNase I footprinting assay. However, the mutation did not alter the pattern of TrpI binding observed in gel retardation experiments. In particular, a more slowly-migrating complex (Complex 2) whose appearance was correlated with TrpI binding to site II was formed equally well on a wild-type or substituted DNA fragment. Based on the mutant phenotypes, we propose that a particular sequence of protein--protein and protein--DNA interactions is required for activation of trpPB by TrpI and InGP.

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Selected References

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

  1. Adhya S., Garges S. Positive control. J Biol Chem. 1990 Jul 5;265(19):10797–10800. [PubMed] [Google Scholar]
  2. Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
  3. Carpousis A. J., Gralla J. D. Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter. Biochemistry. 1980 Jul 8;19(14):3245–3253. doi: 10.1021/bi00555a023. [DOI] [PubMed] [Google Scholar]
  4. Chang M., Crawford I. P. In vitro determination of the effect of indoleglycerol phosphate on the interaction of purified TrpI protein with its DNA-binding sites. J Bacteriol. 1991 Mar;173(5):1590–1597. doi: 10.1128/jb.173.5.1590-1597.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang M., Crawford I. P. The roles of indoleglycerol phosphate and the TrpI protein in the expression of trpBA from Pseudomonas aeruginosa. Nucleic Acids Res. 1990 Feb 25;18(4):979–988. doi: 10.1093/nar/18.4.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang M., Hadero A., Crawford I. P. Sequence of the Pseudomonas aeruginosa trpI activator gene and relatedness of trpI to other procaryotic regulatory genes. J Bacteriol. 1989 Jan;171(1):172–183. doi: 10.1128/jb.171.1.172-183.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crawford I. P. Evolution of a biosynthetic pathway: the tryptophan paradigm. Annu Rev Microbiol. 1989;43:567–600. doi: 10.1146/annurev.mi.43.100189.003031. [DOI] [PubMed] [Google Scholar]
  8. Crawford I. P., Gunsalus I. C. Inducibility of tryptophan synthetase in Pseudomonas putida. Proc Natl Acad Sci U S A. 1966 Aug;56(2):717–724. doi: 10.1073/pnas.56.2.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gao J. G., Gussin G. N. Activation of the trpBA promoter of Pseudomonas aeruginosa by TrpI protein in vitro. J Bacteriol. 1991 Jun;173(12):3763–3769. doi: 10.1128/jb.173.12.3763-3769.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gao J. G., Gussin G. N. RNA polymerases from Pseudomonas aeruginosa and Pseudomonas syringae respond to Escherichia coli activator proteins. J Bacteriol. 1991 Jan;173(1):394–397. doi: 10.1128/jb.173.1.394-397.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Han C. Y., Crawford I. P., Harwood C. S. Up-promoter mutations in the trpBA operon of Pseudomonas aeruginosa. J Bacteriol. 1991 Jun;173(12):3756–3762. doi: 10.1128/jb.173.12.3756-3762.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hawley D. K., Johnson A. D., McClure W. R. Functional and physical characterization of transcription initiation complexes in the bacteriophage lambda OR region. J Biol Chem. 1985 Jul 15;260(14):8618–8626. [PubMed] [Google Scholar]
  13. Henikoff S., Haughn G. W., Calvo J. M., Wallace J. C. A large family of bacterial activator proteins. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6602–6606. doi: 10.1073/pnas.85.18.6602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ho Y. S., Rosenberg M. Characterization of a third, cII-dependent, coordinately activated promoter on phage lambda involved in lysogenic development. J Biol Chem. 1985 Sep 25;260(21):11838–11844. [PubMed] [Google Scholar]
  15. Hochschild A., Irwin N., Ptashne M. Repressor structure and the mechanism of positive control. Cell. 1983 Feb;32(2):319–325. doi: 10.1016/0092-8674(83)90451-8. [DOI] [PubMed] [Google Scholar]
  16. Hwang J. J., Gussin G. N. Interactions between Escherichia coli RNA polymerase and lambda repressor. Mutations in PRM affect repression of PR. J Mol Biol. 1988 Apr 20;200(4):735–739. doi: 10.1016/0022-2836(88)90484-6. [DOI] [PubMed] [Google Scholar]
  17. Johnson A. D., Meyer B. J., Ptashne M. Interactions between DNA-bound repressors govern regulation by the lambda phage repressor. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5061–5065. doi: 10.1073/pnas.76.10.5061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lobell R. B., Schleif R. F. DNA looping and unlooping by AraC protein. Science. 1990 Oct 26;250(4980):528–532. doi: 10.1126/science.2237403. [DOI] [PubMed] [Google Scholar]
  19. Manch J. N., Crawford I. P. Genetic evidence for a positive-acting regulatory factor mediating induction in the tryptophan pathway of Pseudomonas aeruginosa. J Mol Biol. 1982 Mar 25;156(1):67–77. doi: 10.1016/0022-2836(82)90459-4. [DOI] [PubMed] [Google Scholar]
  20. McClure W. R. Rate-limiting steps in RNA chain initiation. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5634–5638. doi: 10.1073/pnas.77.10.5634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. O'Halloran T. V., Frantz B., Shin M. K., Ralston D. M., Wright J. G. The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex. Cell. 1989 Jan 13;56(1):119–129. doi: 10.1016/0092-8674(89)90990-2. [DOI] [PubMed] [Google Scholar]
  22. Ren Y. L., Garges S., Adhya S., Krakow J. S. Cooperative DNA binding of heterologous proteins: evidence for contact between the cyclic AMP receptor protein and RNA polymerase. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4138–4142. doi: 10.1073/pnas.85.12.4138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Summers A. O. Organization, expression, and evolution of genes for mercury resistance. Annu Rev Microbiol. 1986;40:607–634. doi: 10.1146/annurev.mi.40.100186.003135. [DOI] [PubMed] [Google Scholar]
  26. Wek R. C., Hatfield G. W. Transcriptional activation at adjacent operators in the divergent-overlapping ilvY and ilvC promoters of Escherichia coli. J Mol Biol. 1988 Oct 5;203(3):643–663. doi: 10.1016/0022-2836(88)90199-4. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]

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