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Volume 272, Number 6, Issue of February 7, 1997 pp. 3487-3494
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Recognition of the -10 Promoter Sequence by a Partial Polypeptide of sigma 70 in Vitro

(Received for publication, October 9, 1996, and in revised form, November 29, 1996)

Alicia J. Dombroski

From the Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas 77030

Promoter recognition by RNA polymerase depends upon its ability to bind to specific DNA sequences. The sigma (sigma ) subunit provides selectivity for transcription initiation by interacting with the -10 and -35 elements of promoter DNA. Suppressor mutations in sigma  factor that compensate for specific "down" substitutions in the promoter have demonstrated that sigma  factor recognizes certain base pairs of the promoter. Since these suppressors were only identified for changes at the -12 and -11 positions of the -10 element (<UNL>TA</UNL>TAAT), the role of the other base pairs of this region in specifying recognition by sigma  factor remained unclear. Using a partial polypeptide of sigma 70 carrying regions 2-4, this report shows that the first three positions of the -10 element (-12, -11, -10) are important for sigma  factor alone to recognize and bind to duplex DNA. The sigma  polypeptide also binds to an "extended -10" promoter, even without a -35 element. A mismatch bubble from -10 to +3 is bound regardless of the sequence within the bubble, or the presence or absence of a -35 element. Unexpectedly, binding to a mismatch bubble that lacks a -35 hexamer is sensitive to the identity of the -11 position, but not the -12 position.


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