Roles of Clamp Closing and Allosteric Effects of Discriminator and Upstream Interactions on Downstream Elements in Stabilizing an E. coli RNA Polymerase-Promoter Open Complex

Abstract

E. coli RNA polymerase (2{beta}{beta}{omega}{sigma}70) forms stable open complexes (OC) at {lambda}PR and T7A1 promoters at 37 {degrees}C at similar rates but with very different lifetimes. To probe the origins of these differences, we determine OC lifetimes for full-length (FL) and downstream-truncated (DT) {lambda}PR and T7A1 promoter variants with eight combinations of discriminators and upstream elements. We find the discriminator is the major determinant of OC lifetime, while upstream elements modulate the discriminator effect. The very different lifetimes of these stable OC arise primarily from differences in [Formula], the free energy change for converting I2 (the initial open intermediate) to stable OC. [Formula] is twice as favorable for {lambda}PR-discriminator variants as for T7A1-discriminator variants. Truncation at +6 (DT+6) eliminates most differences in [Formula], destabilizing {lambda}PR-discriminator variants without affecting T7A1-discriminator variants. All DT+6 OC are much more stable and longer-lived than I2. Urea greatly destabilizes OC, primarily affecting RNAP interactions with +6 and upstream DNA. From these findings we deduce that stabilization of I2 involves closing the {beta}-clamp with extensive coupled folding to contact the region of promoter DNA from the transcription start-site to +6. Lifetime differences between DT+6, DT+12 and FL promoters allow dissection of contributions to OC stability from interactions of regions of the downstream duplex with downstream mobile elements (DME; {beta}-lobe, {beta}-clamp, {beta}-jaw). We propose an allosteric network by which differences in discriminator and -10 sequence are sensed by {sigma}1.2 and {beta}-gate loop and transmitted to {sigma}1.1 and {beta}-lobe to affect DME-duplex interactions that determine OC lifetime.