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J. Biol. Chem., Vol. 276, Issue 23, 19836-19844, June 8, 2001

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Intrinsic DNA Distortion of the Bacteriophage Mu momP1 Promoter Is a Negative Regulator of Its Transcription
A NOVEL MODE OF REGULATION OF TOXIC GENE EXPRESSION^*

Shashwati Basak^§, Lars Olsen^¶, Stanley Hattman^¶, and Valakunja Nagaraja

From the  Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India and the ^¶ Department of Biology, University of Rochester, Rochester, New York 14627

The momP1 promoter of the bacteriophage Mu mom operon is an example of a weak promoter. It contains a 19-base pair suboptimal spacer between the 35 (ACCACA) and 10 (TAGAAT) hexamers. Escherichia coli RNA polymerase is unable to bind to momP1 on its own. DNA distortion caused by the presence of a run of six T nucleotides overlapping the 5' end of the 10 element might prevent RNA polymerase 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 DNase I revealed distinct differences in cleavage patterns among the various mutants; in addition, compared with the wild type, the mutants showed an increase (variable) 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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