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BIOCHEMISTRY
Sequence-independent upstream DNA–CTD interactions strongly stimulate Escherichia coli RNA polymerase-lacUV5 promoter association

Wilma Ross, and Richard L. Gourse ^*

Department of Bacteriology, University of Wisconsin, 420 Henry Mall, Madison, WI 53706

Edited by E. Peter Geiduschek, University of California at San Diego, La Jolla, CA and approved November 18, 2004 (received for review August 9, 2004)

The C-terminal domains of the two -subunits (CTD) in Escherichia coli RNA polymerase (RNAP) recognize specific sequences called UP elements in some promoters. These interactions can increase transcription dramatically. Previously, effects of upstream DNA–CTD interactions on transcription were quantified relative to control promoters with nonspecific DNA sequences substituted for UP elements. However, contributions of nonspecific upstream DNA–CTD interactions to promoter activity have not been evaluated extensively. Here, we examine effects of removal of CTD, upstream promoter DNA, or both on the rate of open-complex formation with promoters that lack UP elements. Deletion of CTD decreased the composite second-order association rate constant, k_a, of RNAP for the lacUV5 promoter by 10-fold. Much of this effect was attributable to a decrease in the isomerization rate constant, k₂. Removal of promoter DNA upstream of the -35 element also decreased both k_a and k₂ 10-fold. Upstream DNA extending approximately to base pair -100 was sufficient for maximal association rates of wild-type RNAP with lacUV5 promoter fragments. The CTD and upstream DNA did not affect dissociation rates from the open complex. We suggest that sequence-independent upstream DNA interactions with CTD are major contributors to initiation at many (or all) promoters (not merely promoters containing UP elements) and that these interactions facilitate isomerization events occurring well downstream of the -binding sites. In addition to highlighting the functional importance of nonspecific protein–DNA interactions, these results suggest also that UP element–CTD interactions play an even larger role in transcription initiation than appreciated previously.

-subunit | nonspecific interactions | transcription initiation | isomerization

This paper was submitted directly (Track II) to the PNAS office.

Abbreviation: RNAP, RNA polymerase.

^* To whom correspondence should be addressed. E-mail: rgourse{at}bact.wisc.edu.

© 2005 by The National Academy of Sciences of the USA

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