Open AccessThe effects of upstream DNA on open complex formation by Escherichia coli RNA polymerase
Author(s) -
Caroline A. Davis,
W. Michael,
M. Thomas Record,
Ruth M. Saecker
Publication year - 2004
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.0405779102
Subject(s) - transcription bubble , dna footprinting , rna polymerase , transcription (linguistics) , dna , polymerase , biology , base pair , microbiology and biotechnology , footprinting , dna clamp , coding strand , biophysics , abortive initiation , chemistry , promoter , escherichia coli , rna , biochemistry , gene , rna dependent rna polymerase , reverse transcriptase , gene expression , linguistics , philosophy , base sequence
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 lambdaP(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. KMnO(4) 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.