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Originally published In Press as doi:10.1074/jbc.M011790200 on March 6, 2001

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 BasakDagger §, Lars Olsen, Stanley Hattman, and Valakunja NagarajaDagger ||

From the Dagger  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.

* This work was supported by a grant from the Department of Science and Technology, Government of India (to V. N.) and by United States Public Health Service Grant GM29227 from the National Institutes of Health (to S. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a Senior Research Fellowship from the University Grants Commission, Government of India.

|| To whom correspondence should be addressed: Dept. of Microbiology and Cell Biology, Indian Inst. of Science, Bangalore 560 012, India. Tel: 80-360-0668 or 80-309-2598; Fax: 80-360-2697; E-mail: vraj@mcbl.iisc.ernet.in.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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