Transcription initiation is a highly regulated step of gene expression. Here, we discuss the series of large conformational changes set in motion by initial specific binding of bacterial RNA polymerase (RNAP) to promoter DNA and their relevance for regulation. Bending and wrapping of the upstream duplex facilitates bending of the downstream duplex into the active site cleft, nucleating opening of 13 bp in the cleft. The rate-determining opening step, driven by binding free energy, forms an unstable open complex, probably with the template strand in the active site. At some promoters, this initial open complex is greatly stabilized by rearrangements of the discriminator region between the −10 element and +1 base of the nontemplate strand and of mobile in-cleft and downstream elements of RNAP. The rate of open complex formation is regulated by effects on the rapidly-reversible steps preceding DNA opening, while open complex lifetime is regulated by effects on the stabilization of the initial open complex. Intrinsic DNA opening-closing appears less regulated. This noncovalent mechanism and its regulation exhibit many analogies to mechanisms of enzyme catalysis.

To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation by Escherichia coli RNA polymerase (RNAP; α 2 ββ'ωσ 70 ), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λP R and T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of longlived λP R -discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λP R -discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles.RNA polymerase | open complex lifetime | transcription initiation | abortive RNA | hybrid length M any facets of transcription initiation by E. coli RNA polymerase (RNAP; α 2 ββ′ωσ 70 ) have been elucidated, but significant questions remain about the mechanism or mechanisms by which initial transcribing complexes (ITC) with a short RNA-DNA hybrid decide to advance and escape from the promoter to enter elongation mode, or, alternately, to stall, release their short RNA, and reinitiate (abortive cycling). For RNAP to escape, its sequencespecific interactions with promoter DNA in the binary open complex (OC) must be overcome.The open regions of promoter DNA in the binary OC are the −10 region (six residues, with specific interactions between σ 2.2 and the nontemplate strand), the discriminator region (typically six to eight residues with no consensus sequence, the upstream end of which interacts with σ 1.2 ), and the transcription start site (TSS, +1) and adjacent residue (+2), which are in the active site of RNAP (Table 1). The interactions involving and directed by the six-residue λP R discriminator make its OC longlived and highly stable (1). A six-residue discriminator allows the OC to form without deforming (prescrunching) either open discriminator strand (2). Less extensive interactions involving and directed...

In transcription initiation by Escherichia coli RNA polymerase (RNAP), initial binding to promoter DNA triggers large conformational changes, bending downstream duplex DNA into the RNAP cleft and opening 13 base pairs to form a short-lived open intermediate (I2). Subsequent conformational changes increase lifetimes of λPR and T7A1 open complexes (OC) by >105-fold and >102-fold, respectively. OC lifetime is a target for regulation. To characterize late conformational changes, we determine effects on OC dissociation kinetics of deletions in RNAP mobile elements σ70 region 1.1 (σ1.1), β’ jaw and β’ sequence insertion 3 (SI3). In very stable OC formed by WT RNAP with λPR (RPO) and by △σ1.1 RNAP with λPR or T7A1 we conclude that downstream duplex DNA is bound to the jaw in an assembly with SI3, and bases −4 to +2 of the nontemplate strand discriminator region are stably bound in a positively-charged track in the cleft. We deduce that polyanionic σ1.1 destabilizes OC by competing for binding sites in the cleft and on the jaw with the polyanionic discriminator strand and downstream duplex, respectively. Examples of σ1.1-destabilized OC are the final T7A1 OC and the λPR I3 intermediate OC. Deleting σ1.1 and either β’ jaw or SI3 equalizes OC lifetimes for λPR and T7A1. DNA closing rates are similar for both promoters and all RNAP variants. We conclude that late conformational changes that stabilize OC, like early ones that bend the duplex into the cleft, are primary targets of regulation, while the intrinsic DNA opening-closing step is not.

Differences in kinetics of transcription initiation by RNA polymerase (RNAP) at different promoters tailor the pattern of gene expression to cellular needs. After initial binding, large conformational changes occur in promoter DNA and RNAP to form initiation-capable complexes. To understand the mechanism and regulation of transcription initiation, the nature and sequence of these conformational changes must be determined. Escherichia coli RNAP uses binding free energy to unwind and separate 13 base pairs of λPR promoter DNA to form the unstable open intermediate I2, which rapidly converts to much more stable open complexes (I3, RPo). Conversion of I2 to RPo involves folding/assembly of several mobile RNAP domains on downstream duplex DNA. Here, we investigate effects of a 42-residue deletion in the mobile β’ jaw (ΔJAW) and truncation of promoter DNA beyond +12 (DT+12) on the steps of initiation. We find that in stable ΔJAW open complexes the downstream boundary of hydroxyl radical protection shortens by 5–10 base pairs, as compared to wild-type (WT) complexes. Dissociation kinetics of open complexes formed with ΔJAW RNAP and/or DT+12 DNA resemble those deduced for the structurally-uncharacterized intermediate I3. Overall rate constants (ka) for promoter binding and DNA opening by ΔJAW RNAP are much smaller than for WT RNAP. Values of ka for WT RNAP with DT+12 and full-length λPR are similar, though contributions of binding and isomerization steps differ. Hence, the jaw plays major roles both early and late in RPo formation, while downstream DNA functions primarily as the assembly platform after DNA opening.

The catalytic activity of many protein kinases is controlled by conformational changes of a conserved Asp-Phe-Gly (DFG) motif. We used an infrared probe to track the DFG motif of the mitotic kinase Aurora A (AurA) and found that allosteric activation by the spindle-associated protein Tpx2 involves an equilibrium shift towards the active DFG-In state. Förster resonance energy transfer experiments show that the activation loop undergoes a nanometer-scale movement that is tightly coupled to the DFG equilibrium. Tpx2 further activates AurA by stabilizing a water-mediated allosteric network that links the C-helix to the active site through an unusual polar residue in the regulatory spine. The polar spine residue and water network of AurA are essential for phosphorylation-driven activation, but an alternative form of the water network found in related kinases can support Tpx2-driven activation, suggesting that variations in the water-mediated hydrogen bond network mediate regulatory diversification in protein kinases.