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. 1996 Mar;178(6):1614–1622. doi: 10.1128/jb.178.6.1614-1622.1996

Analysis of CRP-CytR interactions at the Escherichia coli udp promoter.

I Brikun 1, K Suziedelis 1, O Stemmann 1, R Zhong 1, L Alikhanian 1, E Linkova 1, A Mironov 1, D E Berg 1
PMCID: PMC177846  PMID: 8626289

Abstract

Multiprotein complexes regulate the transcription of certain bacterial genes in a sensitive, physiologically responsive manner. In particular, the transcription of genes needed for utilization of nucleosides in Escherichia coli is regulated by a repressor protein, CytR, in concert with the cyclic AMP (cAMP) activated form of cAMP receptor protein (CRP). We studied this regulation by selecting and characterizing spontaneous constitutive mutations in the promoter of the udp (uridine phosphorylase) gene, one of the genes most strongly regulated by CytR. We found deletions, duplications, and point mutations that affect key regulatory sites in the udp promoter, insertion sequence element insertions that activated cryptic internal promoters or provided new promoters, and large duplications that may have increased expression by udp gene amplification. Unusual duplications and deletions that resulted in constitutive udp expression that depended on the presence of CytR were also found. Our results support the model in which repression normally involves the binding of CytR to cAMP-CRP to form a complex which binds to specific sites in the udp promoter, without direct interaction between CytR protein and a specific operator DNA sequence, and in which induction by specific inducer cytidine involves dissociation of CytR from cAMP-CRP and the RNA polymerase interaction with cAMP-CRP bound to a site upstream of then transcription start point. The stimulation of udp expression by CytR in certain mutants may reflect its stabilization of cAMP-CRP binding to target DNA and illustrates that only modest evolutionary changes could allow particular multiprotein complexes to serve as either repressors or transcriptional activators.

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Selected References

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  1. Adhya S. Multipartite genetic control elements: communication by DNA loop. Annu Rev Genet. 1989;23:227–250. doi: 10.1146/annurev.ge.23.120189.001303. [DOI] [PubMed] [Google Scholar]
  2. Barbier C. S., Short S. A. Amino acid substitutions in the CytR repressor which alter its capacity to regulate gene expression. J Bacteriol. 1992 May;174(9):2881–2890. doi: 10.1128/jb.174.9.2881-2890.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brikun I., Suziedelis K., Berg D. E. DNA sequence divergence among derivatives of Escherichia coli K-12 detected by arbitrary primer PCR (random amplified polymorphic DNA) fingerprinting. J Bacteriol. 1994 Mar;176(6):1673–1682. doi: 10.1128/jb.176.6.1673-1682.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chapon C., Kolb A. Action of CAP on the malT promoter in vitro. J Bacteriol. 1983 Dec;156(3):1135–1143. doi: 10.1128/jb.156.3.1135-1143.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dombroski A. J., Walter W. A., Record M. T., Jr, Siegele D. A., Gross C. A. Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA. Cell. 1992 Aug 7;70(3):501–512. doi: 10.1016/0092-8674(92)90174-b. [DOI] [PubMed] [Google Scholar]
  6. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gaston K., Bell A., Kolb A., Buc H., Busby S. Stringent spacing requirements for transcription activation by CRP. Cell. 1990 Aug 24;62(4):733–743. doi: 10.1016/0092-8674(90)90118-x. [DOI] [PubMed] [Google Scholar]
  8. Hendrickson W., Flaherty C., Molz L. Sequence elements in the Escherichia coli araFGH promoter. J Bacteriol. 1992 Nov;174(21):6862–6871. doi: 10.1128/jb.174.21.6862-6871.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Holst B., Søgaard-Andersen L., Pedersen H., Valentin-Hansen P. The cAMP-CRP/CytR nucleoprotein complex in Escherichia coli: two pairs of closely linked binding sites for the cAMP-CRP activator complex are involved in combinatorial regulation of the cdd promoter. EMBO J. 1992 Oct;11(10):3635–3643. doi: 10.1002/j.1460-2075.1992.tb05448.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Keilty S., Rosenberg M. Constitutive function of a positively regulated promoter reveals new sequences essential for activity. J Biol Chem. 1987 May 5;262(13):6389–6395. [PubMed] [Google Scholar]
  11. Krishnan B. R., Kersulyte D., Brikun I., Berg C. M., Berg D. E. Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones. Nucleic Acids Res. 1991 Nov 25;19(22):6177–6182. doi: 10.1093/nar/19.22.6177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Mironov A. S., Smirnov Iu V., Sukhodolets V. V. Dal'neishee izuchenie prirody fenotipicheskikh reversii u deletsionnhykh mutantov po timidinfosforilaze Escherichia coli K-12. Genetika. 1975 Apr;11(4):97–105. [PubMed] [Google Scholar]
  14. Mulligan M. E., McClure W. R. Analysis of the occurrence of promoter-sites in DNA. Nucleic Acids Res. 1986 Jan 10;14(1):109–126. doi: 10.1093/nar/14.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. O'Neill M. C. Escherichia coli promoters. I. Consensus as it relates to spacing class, specificity, repeat substructure, and three-dimensional organization. J Biol Chem. 1989 Apr 5;264(10):5522–5530. [PubMed] [Google Scholar]
  16. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  17. Pedersen H., Søgaard-Andersen L., Holst B., Valentin-Hansen P. Heterologous cooperativity in Escherichia coli. The CytR repressor both contacts DNA and the cAMP receptor protein when binding to the deoP2 promoter. J Biol Chem. 1991 Sep 25;266(27):17804–17808. [PubMed] [Google Scholar]
  18. Ponnambalam S., Chan B., Busby S. Functional analysis of different sequence elements in the Escherichia coli galactose operon P2 promoter. Mol Microbiol. 1988 Mar;2(2):165–172. doi: 10.1111/j.1365-2958.1988.tb00018.x. [DOI] [PubMed] [Google Scholar]
  19. RAZZELL W. E., KHORANA H. G. Purification and properties of a pyrimidine deoxyriboside phosphorylase from Escherichia coli. Biochim Biophys Acta. 1958 Jun;28(3):562–566. doi: 10.1016/0006-3002(58)90519-5. [DOI] [PubMed] [Google Scholar]
  20. Rasmussen P. B., Søgaard-Andersen L., Valentin-Hansen P. Identification of the nucleotide sequence recognized by the cAMP-CRP dependent CytR repressor protein in the deoP2 promoter in E. coli. Nucleic Acids Res. 1993 Feb 25;21(4):879–885. doi: 10.1093/nar/21.4.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Søgaard-Andersen L., Valentin-Hansen P. Protein-protein interactions in gene regulation: the cAMP-CRP complex sets the specificity of a second DNA-binding protein, the CytR repressor. Cell. 1993 Nov 5;75(3):557–566. doi: 10.1016/0092-8674(93)90389-8. [DOI] [PubMed] [Google Scholar]
  22. Ushida C., Aiba H. Helical phase dependent action of CRP: effect of the distance between the CRP site and the -35 region on promoter activity. Nucleic Acids Res. 1990 Nov 11;18(21):6325–6330. doi: 10.1093/nar/18.21.6325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  24. Valentin-Hansen P., Holst B., Søgaard-Andersen L., Martinussen J., Nesvera J., Douthwaite S. R. Design of cAMP-CRP-activated promoters in Escherichia coli. Mol Microbiol. 1991 Feb;5(2):433–437. doi: 10.1111/j.1365-2958.1991.tb02126.x. [DOI] [PubMed] [Google Scholar]
  25. Valentin-Hansen P., Larsen J. E., Højrup P., Short S. A., Barbier C. S. Nucleotide sequence of the CytR regulatory gene of E. coli K-12. Nucleic Acids Res. 1986 Mar 11;14(5):2215–2228. doi: 10.1093/nar/14.5.2215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Valentin-Hansen P. Tandem CRP binding sites in the deo operon of Escherichia coli K-12. EMBO J. 1982;1(9):1049–1054. doi: 10.1002/j.1460-2075.1982.tb01295.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Walton L., Richards C. A., Elwell L. P. Nucleotide sequence of the Escherichia coli uridine phosphorylase (udp) gene. Nucleic Acids Res. 1989 Aug 25;17(16):6741–6741. doi: 10.1093/nar/17.16.6741. [DOI] [PMC free article] [PubMed] [Google Scholar]

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