Evidence for Stepwise Disruption of E. coli RNA Polymerase: λP_R Promoter Contacts and Duplex Formation in...

To initiate transcription, the σ⁷⁰ subunit of Escherichia coli RNA polymerase (RNAP) binds duplex promoter DNA regions upstream of the transcription start site (TSS). Then σ⁷⁰ and core RNAP bind and separate the individual DNA strands to open 13 base pairs, including TSS, forming an open complex (OC) that initiates upon binding NTP. To escape the promoter, RNAP uses translocation stress to break RNAP-promoter and σ⁷⁰-core contacts, allowing duplex formation and releasing σ⁷⁰. Here, to determine the timing and mechanism of contact disruption and duplex formation in promoter escape, we measure and interpret the effects of urea and glycine betaine (GB) on rates of individual nucleotide-incorporation steps at the λP_R promoter. These solutes report on step-by-step changes in the amount and type of water-accessible surface area (ASA). We empirically separate contributions to solute effects from contact disruption/duplex formation and from NTP-binding/catalysis/translocation. This separation reveals that contacts are disrupted stepwise throughout mid- and late-initiation (5-mer to 11-mer synthesis). Overall net effects of urea (favorable) and GB (unfavorable) on contact disruption/duplex formation are large in magnitude and well-predicted from OC structures and solute-interaction strengths from model compound data. Comparing predicted and observed solute effects, we deduce that contact disruption/duplex formation proceeds from downstream to upstream. RNAP contacts with open strands break in steps 5-9, allowing duplex formation, while σ⁷⁰ contacts with upstream duplex DNA and core RNAP break in steps 10 and 11, allowing σ⁷⁰ release and promoter escape. These findings validate the stepwise mechanism of contact disruption and demonstrate the power of solutes in mechanistic studies.