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Papers In Press, published online ahead of print March 6, 2001
J. Biol. Chem, 10.1074/jbc.M011790200
Submitted on December 29, 2000
Revised on March 6, 2001
Accepted on March 5, 2001
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Karnataka 560012
Corresponding Author: vraj{at}mcbl.iisc.ernet.in
The momP1 promoter of the bacteriophage Mu mom operon is an example of a weak promoter. It contains a 19 bp suboptimal spacer between the -35 (ACCACA) and -10 (TAGAAT) hexamers. Escherichia coli RNA polymerase (RNAP) is unable to bind to momP1 on its own. DNA distortion, due to the presence of a run of six 'T' nucleotides overlapping the 5' end of the -10 element, might prevent RNAP from binding to momP1. To investigate the influence of the T6-run on momP1 expression, defined substitution mutations were introduced by site-directed mutagenesis. In vitro probing experiments with copper phenanthroline [(OP)2Cu] and DNaseI revealed distinct differences in cleavage patterns among the various mutants; in addition, compared to the wild type, the mutants showed (variable) increase in momP1 promoter activity in vivo. Promoter strength analyses were in agreement with the ability of these mutants to form open complexes as well as to produce momP1-specific transcripts. No significant role is attributed to the overlapping and divergently organized promoter, momP2, in the expression of momP1 activity, as determined by promoter disruption analysis. These data support the view that an intrinsic DNA distortion in the spacer region of momP1 acts in cis as a negative element in mom operon transcription. This is a novel mechanism of regulation of toxic gene expression.
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