Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1991 Dec 25;19(24):6705–6712. doi: 10.1093/nar/19.24.6705

The role of two surface exposed loops in transcription activation by the Escherichia coli CRP and FNR proteins.

R Williams 1, A Bell 1, G Sims 1, S Busby 1
PMCID: PMC329298  PMID: 1762901

Abstract

We have investigated a number of mutations that alter the ability of the E. coli transcription factors CRP and FNR to activate transcription. In CRP, some mutations at position 159 (H159L, H159I and delta 159) prevent transcription activation at a number of naturally-occurring and semi-synthetic CRP-dependent promoters. We suggest that some feature of the surface-exposed turn around residue 159 is recognised by RNA polymerase during transcription activation at these promoters. Mutations at position 52 increase CRP activity and reverse the effects of H159L and delta 159, most likely by creating a new contact with RNA polymerase. However this new contact only gives increased expression when the CRP binding site is located 41 1/2 base pairs upstream of the transcription start site and fails to reverse the effects of H159L and delta 159 at promoters where the CRP site is located further upstream. To explain our results we propose that the two surface-exposed turns around residues 52 and 159 contain elements that are potential RNA polymerase docking sites: in the CRP dimer these two active patches are located on adjacent faces of different subunits. FNR, a related transcription activator, contains amino acid sequences homologous to the CRP sequence around position 52. Mutations in this zone (from residues 81-88 in FNR) reduce expression from an FNR-dependent promoter without stopping FNR binding to its target. This defines a patch on FNR, which is homologous to the CRP surface-exposed loop around position 52, which is involved in transcription activation, most likely by contacting RNA polymerase.

Full text

PDF
6705

Selected References

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

  1. Aiba H. Autoregulation of the Escherichia coli crp gene: CRP is a transcriptional repressor for its own gene. Cell. 1983 Jan;32(1):141–149. doi: 10.1016/0092-8674(83)90504-4. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bell A., Gaston K., Williams R., Chapman K., Kolb A., Buc H., Minchin S., Williams J., Busby S. Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription. Nucleic Acids Res. 1990 Dec 25;18(24):7243–7250. doi: 10.1093/nar/18.24.7243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Busby S., Buc H. Positive regulation of gene expression by cyclic AMP and its receptor protein in Escherichia coli. Microbiol Sci. 1987 Dec;4(12):371–375. [PubMed] [Google Scholar]
  5. Busby S., Spassky A., Chan B. RNA polymerase makes important contacts upstream from base pair -49 at the Escherichia coli galactose operon P1 promoter. Gene. 1987;53(2-3):145–152. doi: 10.1016/0378-1119(87)90002-3. [DOI] [PubMed] [Google Scholar]
  6. Cossart P., Gicquel-Sanzey B. Cloning and sequence of the crp gene of Escherichia coli K 12. Nucleic Acids Res. 1982 Feb 25;10(4):1363–1378. doi: 10.1093/nar/10.4.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eschenlauer A. C., Reznikoff W. S. Escherichia coli catabolite gene activator protein mutants defective in positive control of lac operon transcription. J Bacteriol. 1991 Aug;173(16):5024–5029. doi: 10.1128/jb.173.16.5024-5029.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Green J., Trageser M., Six S., Unden G., Guest J. R. Characterization of the FNR protein of Escherichia coli, an iron-binding transcriptional regulator. Proc Biol Sci. 1991 May 22;244(1310):137–144. doi: 10.1098/rspb.1991.0062. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Kolb A., Busby S., Herbert M., Kotlarz D., Buc H. Comparison of the binding sites for the Escherichia coli cAMP receptor protein at the lactose and galactose promoters. EMBO J. 1983;2(2):217–222. doi: 10.1002/j.1460-2075.1983.tb01408.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lobell R. B., Schleif R. F. AraC-DNA looping: orientation and distance-dependent loop breaking by the cyclic AMP receptor protein. J Mol Biol. 1991 Mar 5;218(1):45–54. doi: 10.1016/0022-2836(91)90872-4. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Schleif R., Hess W., Finkelstein S., Ellis D. Induction kinetics of the L-arabinose operon of Escherichia coli. J Bacteriol. 1973 Jul;115(1):9–14. doi: 10.1128/jb.115.1.9-14.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Schultz S. C., Shields G. C., Steitz T. A. Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees. Science. 1991 Aug 30;253(5023):1001–1007. doi: 10.1126/science.1653449. [DOI] [PubMed] [Google Scholar]
  16. Sharrocks A. D., Green J., Guest J. R. FNR activates and represses transcription in vitro. Proc Biol Sci. 1991 Sep 23;245(1314):219–226. doi: 10.1098/rspb.1991.0113. [DOI] [PubMed] [Google Scholar]
  17. Spassky A., Busby S., Buc H. On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions. EMBO J. 1984 Jan;3(1):43–50. doi: 10.1002/j.1460-2075.1984.tb01759.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Spiro S., Gaston K. L., Bell A. I., Roberts R. E., Busby S. J., Guest J. R. Interconversion of the DNA-binding specificities of two related transcription regulators, CRP and FNR. Mol Microbiol. 1990 Nov;4(11):1831–1838. doi: 10.1111/j.1365-2958.1990.tb02031.x. [DOI] [PubMed] [Google Scholar]
  19. Spiro S., Guest J. R. Regulation and over-expression of the fnr gene of Escherichia coli. J Gen Microbiol. 1987 Dec;133(12):3279–3288. doi: 10.1099/00221287-133-12-3279. [DOI] [PubMed] [Google Scholar]
  20. Spiro S., Roberts R. E., Guest J. R. FNR-dependent repression of the ndh gene of Escherichia coli and metal ion requirement for FNR-regulated gene expression. Mol Microbiol. 1989 May;3(5):601–608. doi: 10.1111/j.1365-2958.1989.tb00207.x. [DOI] [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. 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]
  23. Wu H. M., Crothers D. M. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. doi: 10.1038/308509a0. [DOI] [PubMed] [Google Scholar]
  24. Zhang X. P., Ebright R. H. Substitution of 2 base pairs (1 base pair per DNA half-site) within the Escherichia coli lac promoter DNA site for catabolite gene activator protein places the lac promoter in the FNR regulon. J Biol Chem. 1990 Jul 25;265(21):12400–12403. [PubMed] [Google Scholar]
  25. Zinkel S. S., Crothers D. M. Catabolite activator protein-induced DNA bending in transcription initiation. J Mol Biol. 1991 May 20;219(2):201–215. doi: 10.1016/0022-2836(91)90562-k. [DOI] [PubMed] [Google Scholar]
  26. 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]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES