Clamp Interactions with +3/+6 Duplex and Upstream-to-Downstream Allosteric Effects in Late Steps of Forming a Stable RNA Polymerase-Promoter Open Complex.

Stable 37 °C open complexes (OC) of E. coli RNA polymerase (RNAP) at λP and T7A1 promoters form at similar rates but have very different lifetimes. To understand the downstream interactions responsible for OC lifetime, how promoter sequence directs them and when they form, we report lifetimes of stable OC and unstable late (I) intermediates for promoters with different combinations of λP (L) and T7A1 (T) discriminators, core promoters and UP elements. I lifetimes are similarly short, while stable OC lifetimes differ greatly, determined largely by the discriminator and modulated by core-promoter and UP elements. The free energy change ΔG for I → stable OC is approximately -4 kcal more favorable for L-discriminator than for T-discriminator promoters. Downstream-truncation at +6 (DT+6) greatly destabilizes OC at L-discriminator but not T-discriminator promoters, making all ΔG values similar (approximately -4 kcal). Urea reduces OC lifetime greatly by affecting ΔG. We deduce that urea acts by disfavoring coupled folding of key elements of the β'-clamp, that I is an open-clamp OC, and that clamp-closing in I → stable OC involves coupled folding. Differences in ΔG between downstream-truncated and full-length promoters yield contributions to ΔG from interactions with downstream mobile elements (DME) including β-lobe and β'-jaw, more favorable for L-discriminator than for T-discriminator promoters. We deduce how competition between far-downstream DNA and σ region 1.1 affects ΔG values. We discuss variant-specific ΔG contributions in terms of the allosteric network by which differences in discriminator and -10 sequence are sensed and transmitted downstream to affect DME-duplex interactions in I → stable OC.