Solute probes of conformational changes in open complex (RPo) formation by Escherichia coli RNA polymerase at the lambdaPR promoter: evidence for unmasking of the active site in the isomerization step and for large-scale coupled folding in the subsequent conversion to RPo.
Transcription initiation is a multistep process involving a series of requisite conformational changes in RNA polymerase (R) and promoter DNA (P) that create the open complex (RP(o)). Here, we use the small solutes urea and glycine betaine (GB) to probe the extent and type of surface area changes in the formation of RP(o) between Esigma(70) RNA polymerase and lambdaP(R) promoter DNA. Effects of urea quantitatively reflect changes in amide surface and are particularly well-suited to detect coupled protein folding events. GB provides a qualitative probe for the exposure or burial of anionic surface. Kinetics of formation and dissociation of RP(o) reveal strikingly large effects of the solutes on the final steps of RP(o) formation: urea dramatically increases the dissociation rate constant k(d), whereas GB decreases the rate of dissociation. Formation of the first kinetically significant intermediate I(1) is disfavored in urea, and moderately favored by GB. GB slows the rate-determining step that converts I(1) to the second kinetically significant intermediate I(2); urea has no effect on this step. The most direct interpretation of these data is that recognition of promoter DNA in I(1) involves only limited conformational changes. Notably, the data support the following hypotheses: (1) the negatively charged N-terminal domain of sigma(70) remains bound in the "jaws" of polymerase in I(1); (2) the subsequent rate-determining isomerization step involves ejecting this domain from the jaws, thereby unmasking the active site; and (3) final conversion to RP(o) involves coupled folding of the mobile downstream clamp of polymerase.
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Wayne S
WS
Kontur
Ruth M
RM
Saecker
Caroline A
CA
Davis
Michael W
MW
Capp
M Thomas
MT
Record
D010582:Bacteriophage lambda / Q000235:genetics*; D001622:Betaine / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011487:Protein Conformation / Q000187:drug effects; D012808:Sigma Factor / Q000737:chemistry* / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D029968:Escherichia coli Proteins; D012808:Sigma Factor; C090751:dksA protein, E coli; D001622:Betaine; D014508:Urea; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases