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Journal of Bacteriology, November 2001, p. 6413-6421, Vol. 183, No. 21
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.21.6413-6421.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Bacterial Two-Hybrid Analysis of Interactions between Region 4 of the sigma 70 Subunit of RNA Polymerase and the Transcriptional Regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa

Simon L. Dove and Ann Hochschild*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

Received 3 May 2001/Accepted 6 August 2001

A number of transcriptional regulators mediate their effects through direct contact with the sigma 70 subunit of Escherichia coli RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of sigma 70 that harbors conserved region 4. This region of sigma  contains a putative helix-turn-helix DNA-binding motif that contacts the -35 element of sigma 70-dependent promoters directly. Here we report the use of a recently developed bacterial two-hybrid system to study the interaction between the putative anti-sigma factor Rsd and the sigma 70 subunit of E. coli RNAP. Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of sigma 70 and also that amino acid substitution R596H, within region 4 of sigma 70, weakens this interaction. We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between sigma  and two other regulators shown previously to contact region 4 of sigma 70. We also demonstrated that AlgQ, a homolog of Rsd that positively regulates virulence gene expression in Pseudomonas aeruginosa, can contact the C-terminal region of the sigma 70 subunit of RNAP from this organism. We found that amino acid substitution R600H in sigma 70 from P. aeruginosa, corresponding to the R596H substitution in E. coli sigma 70, specifically weakens the interaction between AlgQ and sigma 70. Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of sigma 70 and probably regulate gene expression through this contact.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115. Phone: (617) 432-1986. Fax: (617) 738-7664. E-mail: ahochschild{at}hms.harvard.edu.

Journal of Bacteriology, November 2001, p. 6413-6421, Vol. 183, No. 21
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.21.6413-6421.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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