Solute Probes of Conformational Changes in Open Complex (RP_o) Formation by Escherichia coli RNA Polymerase at the λP_R Promoter:  Evidence for Unmasking of the Active Site in the Isomerization Step and for Large-Scale Coupled Folding in the Subsequent Conversion to RP_o^†

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 Eσ⁷⁰ RNA polymerase and λP_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 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 RP_o involves coupled folding of the mobile downstream clamp of polymerase.