Nucleic Acids Research, 1992, Vol. 20, No. 11 2777-2784
© 1992
MOLECULAR BIOLOGY |
Functionally distinct RNA polymerase binding sites in the phage Mu mom promoter region
University of Rochester, Department of Biology Rochester, NY 14627, USA
*To whom correspondence should be addressed
Received March 18, 1992. Revised April 27, 1992. Accepted April 27, 1992.
Transcription of the phage Mu com/mom operon is trans-activated by another phage gene product, C, a site-specific DNA binding protein. To gain insight into the mechanism by which C activates transcription, we carried out footprinting analyses of Escherichia coli RNA polymerase (=RNAP) binding to various com-iacZ fusion piasmids. KMnO4-sensitive sites (diagnostic of the melted regions in open-complexes) and DNase I-sensitive sites were located by primer-extension analysis. The results are summarized as follows: (i)In vivo, in the absence of C, RNAP bound in the wild-type (wt) promoter region at a site designated P2; in vitro DNase I-footprinting showed that P2 extends from –74 to –24 with respect to transcription initiation. This overlaps a known strong C-binding site (at –35 to –54). RNAP bound at P2 appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites. (ii) in contrast, when C was present in vivo, RNAP bound in the wt promoter region at a different site, designated P1, located downstream and partially overlapping P2. RNAP bound at P1, also appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites. (iii) Two C-independent mutants, which initiate transcription at the same position as the wt, were also analyzed. In vivo, in the absence of C, RNAP bound mutant tin7 (contains a T to G substitution at –14) predominantly at P1; in vitro DNase I-footprinting showed that P1 extends from –56 to +21. With mutant tIn6 (a 63 base-pair deletion removing P2, as well as part of P1 and the C-binding site from –35 to –54), RNAP bound to P1 independent of C. We conclude that P1 is the ‘functional’ RNAP binding site for mom-transcription initiation, and that C activates transcription by promoting binding at P1, while blocking binding at P2.
+Present address: Centre for Genetic Engineering, Indian Institute of Science, Bangalore-560012, India
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