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 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 â†’ 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 ß'-clamp, that I2 is an open-clamp OC, and that clamp-closing in I2 â†’ 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 ß-lobe and ß'-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 â†’ stable OC.

Step-by-Step Regulation of Productive and Abortive Transcription Initiation by Pyrophosphorolysis.

An understanding of the kinetics and mechanism of bacterial transcription initiation is needed to understand regulation of gene expression and advance fields from antibiotic discovery to promoter design. The step-by-step forward kinetics and mechanism of initiation and RNA-DNA hybrid growth, made irreversible by omitting pyrophosphate (PPi) byproduct, were determined recently for E. coli RNA polymerase (RNAP)-λPR promoter complexes. Strong position-dependences of overall rate constants (kcat/Km analogs) for each nucleotide-addition step were observed because of coupling of hybrid growth to disruption of promoter contacts, bubble closing, and RNAP escape. Here we investigate reversal of these steps (pyrophosphorolysis) at PPi concentrations ([PPi]) found in exponentially-growing cells. We quantify [PPi] effects on the amount and rate of synthesis of long (>10-mer, post-escape) and short (stalled, abortive) RNA to determine how PPi regulates initiation. Physiological [PPi] makes uridine incorporation and some other initiation steps significantly reversible. Physiological [PPi] reduces the fraction of RNAP-promoter complexes that productively initiate and the rate of RNA synthesis per productive complex, while increasing the fraction of complexes that abortively initiate, affecting abortive rates, and shifting the abortive-product distribution to shorter RNAs. Pyrophosphorolysis rates for some initiation complexes are orders of magnitude larger than for removal of the same nucleotide from elongation complexes because of the strong bias toward the pre-translocated state in initiation, and exhibit even stronger dependences on nucleotide identity (pyrimidine ≫ purine). Because cytoplasmic [PPi] is much higher in exponential-phase than stationary-phase cells, these [PPi] effects on initiation rates and amounts of RNA synthesis must be physiologically-relevant.