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

Abstract

Stable 37 °C open complexes (OC) of E. coli RNA polymerase (RNAP) at λPR 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 (I2) intermediates for promoters with different combinations of λPR (L) and T7A1 (T) discriminators, core promoters and UP elements. I2 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 ΔG3o for I2 → 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 ΔG3o values similar (approximately −4 kcal). Urea reduces OC lifetime greatly by affecting ΔG3o. We deduce that urea acts by disfavoring coupled folding of key elements of the β’-clamp, that I2 is an open-clamp OC, and that clamp-closing in I2 → stable OC involves coupled folding. Differences in ΔG3o between downstream-truncated and full-length promoters yield contributions to ΔG3o 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 σ70 region 1.1 affects ΔG3o values. We discuss variant-specific ΔG3o 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 I2 → stable OC.