Real-time Characterization of Transcription Initiation Intermediates for E. coli RNA Polymerase Using Fast Footprinting and Equilibrium and Stopped-flow Fluorescence

The pathway by which E. coli RNA polymerase (RNAP) forms initiation-capable open complexes at the bacteriophage lambda PR promoter involves at least two key intermediates (designated I1, I2). We used equilibrium and time-resolved footprinting and fluorescence assays to characterize these intermediates and to dissect the detailed mechanism of initiation at lambda PR. HO· snapshots show that I 1 forms rapidly (in 0.1 s); however, fast MnO4- footprinting at 19°C reveals no reactivity of any DNA bases in I1, indicating that promoter DNA in the cleft is still duplex.;We report FRET-monitored equilibrium titrations at 2°C where I 1 is the only promoter complex, and at 10, 19 and 37°C to compare FRET effects in open complexes at these temperatures. Both equilibrium FRET measurements on I1 at 2°C and the initial phase of real-time association kinetic experiments at 19°C exhibit large FRET effects, providing compelling evidence for bending and wrapping of upstream and downstream duplex promoter DNA on RNAP in the initial closed intermediate. Our results suggest that upstream wrapping occurs soon after formation of the HO·-detected I1 complex but before base-flipping of -11A and DNA opening in the cleft. We also monitored changes in stopped-flow fluorescence of the sigma70 subunit during transcription initiation at the lambda PR promoter using intrinsic and "beacon" probes. From comparisons of the two assays, we deduce that the two fluorescent exponential phases represent the decay-to-equilibrium formation of a late species of I1 in which the -11 A base is flipped out of the bent duplex; the slow phase represents the conversion of these closed species to open complexes.;These results support the proposal that RNAP is a molecular isomerization machine that, after initial specific binding, first bends the DNA duplex toward the cleft to form a bent closed intermediate I1,B detected by fast HO· footprinting. Subsequent upstream bending and wrapping converts I1,B to I1,W. Next, base flipping converts I1,W to I1,F. I1,F is poised to open in the rate-determining step in the cleft to form the initial open intermediate I2. Finally, assembly of downstream mobile elements on the downstream DNA duplex form the more stable open complexes (I3, RPo), which are also wrapped.