The effects of upstream DNA on open complex formation by Escherichia coli RNA polymerase

Departments of ^*Biochemistry and ^†Chemistry, University of Wisconsin, Madison, WI 53706

Edited by E. Peter Geiduschek, University of California at San Diego, La Jolla, CA (received for review August 6, 2004)

Abstract

Binding of activators to upstream DNA sequences regulates transcription initiation by affecting the stability of the initial RNA polymerase (RNAP)–promoter complex and/or the rate of subsequent conformational changes required to form the open complex (RP_O). Here we observe that the presence of nonspecific upstream DNA profoundly affects an early step in formation of the transcription bubble. Kinetic studies with the λP_R promoter and Escherichia coli RNAP reveal that the presence of DNA upstream of base pair -47 greatly increases the rate of forming RP_O, without significantly affecting its rate of dissociation. We find that this increase is largely due to an acceleration of the rate-limiting step (isomerization) in RP_O formation, a step that occurs after polymerase binds. Footprinting experiments reveal striking structural differences downstream of the transcription start site (+1) in the first kinetically significant intermediate when upstream DNA is present. On the template strand, the DNase I downstream boundary of this early intermediate is +20 when upstream DNA is present but is shortened by approximately two helical turns when upstream DNA beyond -47 is removed. KMnO₄ footprinting reveals an identical initiation bubble (-11 to +2), but unusual reactivity of template strand upstream cytosines (-12, -14, and -15) on the truncated promoter. Based on this work, we propose that early wrapping interactions between upstream DNA and the polymerase exterior strongly affect the events that control entry and subsequent unwinding of the DNA start site in the jaws of polymerase.

Footnotes

↵ ‡ To whom correspondence should be addressed. E-mail: rmsaecker{at}wisc.edu.
Author contributions: C.A.D., M.T.R., and R.M.S. designed research; C.A.D., M.W.C., and R.M.S. performed research; M.W.C. contributed new reagents/analytic tools; C.A.D., M.T.R., and R.M.S. analyzed data; and C.A.D., M.T.R., and R.M.S. wrote the paper
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: RNAP, RNA polymerase; RP_O, open complex; UT, upstream truncated; FL, full length; CR, competitor resistant; t, template; nt, nontemplate; CTD, C-terminal domain; I, kinetically significant intermediate.