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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
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
70 subunit of
Escherichia coli RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of
70 that harbors conserved region 4. This region of
contains a putative helix-turn-helix DNA-binding motif that contacts
the
35 element of
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-
factor
Rsd and the
70 subunit of E. coli RNAP.
Using this system, we found that Rsd can interact with an 86-amino-acid
C-terminal fragment of
70 and also that amino acid
substitution R596H, within region 4 of
70, weakens this
interaction. We demonstrated the specificity of this effect by showing
that substitution R596H does not weaken the interaction between
and
two other regulators shown previously to contact region 4 of
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
70 subunit of RNAP from this organism. We
found that amino acid substitution R600H in
70 from
P. aeruginosa, corresponding to the R596H substitution
in E. coli
70, specifically weakens the
interaction between AlgQ and
70. Taken together, our
findings suggest that Rsd and AlgQ contact similar surfaces of RNAP
present in region 4 of
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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