bi051835v_si_001.pdf (1.71 MB)
Solute Probes of Conformational Changes in Open Complex (RPo) Formation by Escherichia coli RNA Polymerase at the λPR Promoter: Evidence for Unmasking of the Active Site in the Isomerization Step and for Large-Scale Coupled Folding in the Subsequent Conversion to RPo†
journal contributionposted on 2006-02-21, 00:00 authored by Wayne S. Kontur, Ruth M. Saecker, Caroline A. Davis, Michael W. Capp, M. Thomas Record
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 (RPo). 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 RPo between Eσ70 RNA polymerase and λPR 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 RPo reveal strikingly large effects of the solutes on the final steps of RPo formation: urea dramatically increases the dissociation rate constant kd, whereas GB decreases the rate of dissociation. Formation of the first kinetically significant intermediate I1 is disfavored in urea, and moderately favored by GB. GB slows the rate-determining step that converts I1 to the second kinetically significant intermediate I2; urea has no effect on this step. The most direct interpretation of these data is that recognition of promoter DNA in I1 involves only limited conformational changes. Notably, the data support the following hypotheses: (1) the negatively charged N-terminal domain of σ70 remains bound in the “jaws” of polymerase in I1; (2) the subsequent rate-determining isomerization step involves ejecting this domain from the jaws, thereby unmasking the active site; and (3) final conversion to RPo involves coupled folding of the mobile downstream clamp of polymerase.