Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription (original) (raw)

Abstract

The effects of a number of mutations in the E. coli cyclic AMP receptor protein (CRP) have been determined by monitoring the in vivo expression and in vitro open complex formation at two semi-synthetic promoters that are totally CRP-dependent. At one promoter the CRP-binding site is centered around 41.5 base pairs upstream from the transcription start whilst at the other promoter it is 61.5 base pairs upstream. The CRP mutation E171K reduces expression from both promoters whilst H159L renders CRP totally inactive: neither mutation stops CRP binding at either promoter. The mutations K52N and K52Q reverse the effect of H159L and 'reeducate' CRP to activate transcription. CRP carrying both H159L and K52N activates transcription from the promoter with the CRP site at -41.5 better than wild type CRP. In sharp contrast, this doubly changed CRP is totally inactive with respect to the activation of transcription from the promoter carrying the CRP site at -61.5. Our results suggest that CRP can use different contacts and/or conformations during transcription activation at promoters with different architectures.

7243

Images in this article

Selected References

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

  1. Aiba H., Fujimoto S., Ozaki N. Molecular cloning and nucleotide sequencing of the gene for E. coli cAMP receptor protein. Nucleic Acids Res. 1982 Feb 25;10(4):1345–1361. doi: 10.1093/nar/10.4.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aiba H., Hanamura A., Tobe T. Semisynthetic promoters activated by cyclic AMP receptor protein of Escherichia coli. Gene. 1989 Dec 21;85(1):91–97. doi: 10.1016/0378-1119(89)90468-x. [DOI] [PubMed] [Google Scholar]
  3. Aiba H., Nakamura T., Mitani H., Mori H. Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli. EMBO J. 1985 Dec 1;4(12):3329–3332. doi: 10.1002/j.1460-2075.1985.tb04084.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Balbás P., Soberón X., Merino E., Zurita M., Lomeli H., Valle F., Flores N., Bolivar F. Plasmid vector pBR322 and its special-purpose derivatives--a review. Gene. 1986;50(1-3):3–40. doi: 10.1016/0378-1119(86)90307-0. [DOI] [PubMed] [Google Scholar]
  5. Bell A. I., Gaston K. L., Cole J. A., Busby S. J. Cloning of binding sequences for the Escherichia coli transcription activators, FNR and CRP: location of bases involved in discrimination between FNR and CRP. Nucleic Acids Res. 1989 May 25;17(10):3865–3874. doi: 10.1093/nar/17.10.3865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brosius J. Plasmid vectors for the selection of promoters. Gene. 1984 Feb;27(2):151–160. doi: 10.1016/0378-1119(84)90136-7. [DOI] [PubMed] [Google Scholar]
  7. Busby S., Dreyfus M. Segment-specific mutagenesis of the regulatory region in the Escherichia coli galactose operon: isolation of mutations reducing the initiation of transcription and translation. Gene. 1983 Jan-Feb;21(1-2):121–131. doi: 10.1016/0378-1119(83)90154-3. [DOI] [PubMed] [Google Scholar]
  8. Busby S., Kotlarz D., Buc H. Deletion mutagenesis of the Escherichia coli galactose operon promoter region. J Mol Biol. 1983 Jun 25;167(2):259–274. doi: 10.1016/s0022-2836(83)80335-0. [DOI] [PubMed] [Google Scholar]
  9. Chan B., Busby S. Recognition of nucleotide sequences at the Escherichia coli galactose operon P1 promoter by RNA polymerase. Gene. 1989 Dec 14;84(2):227–236. doi: 10.1016/0378-1119(89)90496-4. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Gaston K., Chan B., Kolb A., Fox J., Busby S. Alterations in the binding site of the cyclic AMP receptor protein at the Escherichia coli galactose operon regulatory region. Biochem J. 1988 Aug 1;253(3):809–818. doi: 10.1042/bj2530809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gaston K., Kolb A., Busby S. Binding of the Escherichia coli cyclic AMP receptor protein to DNA fragments containing consensus nucleotide sequences. Biochem J. 1989 Jul 15;261(2):649–653. doi: 10.1042/bj2610649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ghosaini L. R., Brown A. M., Sturtevant J. M. Scanning calorimetric study of the thermal unfolding of catabolite activator protein from Escherichia coli in the absence and presence of cyclic mononucleotides. Biochemistry. 1988 Jul 12;27(14):5257–5261. doi: 10.1021/bi00414a046. [DOI] [PubMed] [Google Scholar]
  14. Göransson M., Forsman P., Nilsson P., Uhlin B. E. Upstream activating sequences that are shared by two divergently transcribed operons mediate cAMP-CRP regulation of pilus-adhesin in Escherichia coli. Mol Microbiol. 1989 Nov;3(11):1557–1565. doi: 10.1111/j.1365-2958.1989.tb00141.x. [DOI] [PubMed] [Google Scholar]
  15. Irwin N., Ptashne M. Mutants of the catabolite activator protein of Escherichia coli that are specifically deficient in the gene-activation function. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8315–8319. doi: 10.1073/pnas.84.23.8315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lodge J., Williams R., Bell A., Chan B., Busby S. Comparison of promoter activities in Escherichia coli and Pseudomonas aeruginosa: use of a new broad-host-range promoter-probe plasmid. FEMS Microbiol Lett. 1990 Jan 15;55(1-2):221–225. doi: 10.1016/0378-1097(90)90199-z. [DOI] [PubMed] [Google Scholar]
  17. Morita T., Shigesada K., Kimizuka F., Aiba H. Regulatory effect of a synthetic CRP recognition sequence placed downstream of a promoter. Nucleic Acids Res. 1988 Aug 11;16(15):7315–7332. doi: 10.1093/nar/16.15.7315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sibanda B. L., Blundell T. L., Thornton J. M. Conformation of beta-hairpins in protein structures. A systematic classification with applications to modelling by homology, electron density fitting and protein engineering. J Mol Biol. 1989 Apr 20;206(4):759–777. doi: 10.1016/0022-2836(89)90583-4. [DOI] [PubMed] [Google Scholar]
  19. Stoker N. G., Fairweather N. F., Spratt B. G. Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene. 1982 Jun;18(3):335–341. doi: 10.1016/0378-1119(82)90172-x. [DOI] [PubMed] [Google Scholar]
  20. Søgaard-Andersen L., Møllegaard N. E., Douthwaite S. R., Valentin-Hansen P. Tandem DNA-bound cAMP-CRP complexes are required for transcriptional repression of the deoP2 promoter by the CytR repressor in Escherichia coli. Mol Microbiol. 1990 Sep;4(9):1595–1601. [PubMed] [Google Scholar]
  21. 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]
  22. Weber I. T., Steitz T. A. Model of specific complex between catabolite gene activator protein and B-DNA suggested by electrostatic complementarity. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3973–3977. doi: 10.1073/pnas.81.13.3973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Weber I. T., Steitz T. A. Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution. J Mol Biol. 1987 Nov 20;198(2):311–326. doi: 10.1016/0022-2836(87)90315-9. [DOI] [PubMed] [Google Scholar]
  24. de Crombrugghe B., Busby S., Buc H. Cyclic AMP receptor protein: role in transcription activation. Science. 1984 May 25;224(4651):831–838. doi: 10.1126/science.6372090. [DOI] [PubMed] [Google Scholar]