Journal of Bacteriology, September 1999, p. 5185-5192, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045
Received 17 March 1999/Accepted 15 June 1999
RhaS, an AraC family protein, activates rhaBAD
transcription by binding to rhaI, a site consisting of two
17-bp inverted repeat half-sites. In this work, amino acids in RhaS
that make base-specific contacts with rhaI were identified.
Sequence similarity with AraC suggested that the first contacting motif
of RhaS was a helix-turn-helix. Assays of rhaB-lacZ
activation by alanine mutants within this potential motif indicated
that residues 201, 202, 205, and 206 might contact rhaI.
The second motif was identified based on the hypothesis that a region
of especially high amino acid similarity between RhaS and RhaR (another
AraC family member) might contact the nearly identical DNA sequences in
one major groove of their half-sites. We first made targeted, random
mutations and then made alanine substitutions within this region of
RhaS. Our analysis identified residues 247, 248, 250, 252, 253, and 254 as potentially important for DNA binding. A genetic loss-of-contact
approach was used to identify whether any of the RhaS amino acids in
the first or second contacting motif make base-specific DNA contacts. In motif 1, we found that Arg202 and Arg206 both make specific contacts
with bp
65 and
67 in rhaI1, and that Arg202
contacts
46 and Arg206 contacts
48 in
rhaI2. In motif 2, we found that Asp250 and
Asn252 both contact the bp
79 in rhaI1.
Alignment with the recently crystallized MarA protein suggest that both RhaS motifs are likely helix-turn-helix DNA-binding motifs.
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