Emily Ruff

Initial Events in Bacterial Transcription Initiation

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.

Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase

Significance The enzyme RNA polymerase (RNAP) transcribes DNA genetic information into RNA. Regulation of transcription occurs largely in initiation; these regulatory mechanisms must be understood. Lifetimes of transcription-capable RNAP-promoter open complexes (OCs) vary greatly, dictated largely by the DNA discriminator region, but the significance of OC lifetime for regulation was unknown. We observe that a significantly longer RNA:DNA hybrid is synthesized before RNAP escapes from long-lived λPR-promoter OCs as compared with shorter-lived T7A1 promoter OCs. We quantify the free energy needed to overcome OC stability and allow escape from the promoter and elongation of the nascent RNA, and thereby predict escape points for ribosomal (rrnB P1) and lacUV5 promoters. Longer-lived OCs produce longer abortive RNAs, which likely have specific regulatory roles. To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation by Escherichia coli RNA polymerase (RNAP; α2ββ’ωσ70), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λPR 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 long-lived λPR-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, λPR-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.

Using solutes and kinetics to probe large conformational changes in the steps of transcription initiation.

Small solutes are useful probes of large conformational changes in RNA polymerase-promoter interactions and other biopolymer processes. In general, a large effect of a solute on an equilibrium constant (or rate constant) indicates a large change in water-accessible biopolymer surface area in the corresponding step (or transition state), resulting from conformational changes, interface formation, or both. Here, we describe nitrocellulose filter binding assays from series used to determine the urea dependence of open complex formation and dissociation with Escherichia coli RNA polymerase and phage λPR promoter DNA. Then, we describe the subsequent data analysis and interpretation of these solute effects.

Large Effects of Discriminator Exchanges on the RNA Polymerase-Promoter Open Complex Structure, Lifetime and Transcription Initiation Patterns

Escherichia coli RNA polymerase holoenzyme (RNAP; consisting of α2ββ’ω + σ70 subunits) is the molecular machine of transcription. This machinery is set in motion by initial recognition of −35 and −10 regions of linear promoter DNA by σ70 and of the UP element region by the two flexibly-tethered α-CTDs. A series of large conformational changes in both RNAP and DNA, driven by binding free energy, bend the downstream duplex into the active site cleft and open 13 base pairs including the −10 and discriminator regions and the transcription start site (+1). Subsequently at some promoters the initial unstable (lifetime 1 s) open complex is stabilized by a network of interactions involving the discriminator DNA, σ70 region 1.1, and downstream mobile elements (DME) of RNAP. These increase open complex lifetime to 6 minutes for T7A1 promoter and 17 hrs for the λPR promoter. An important but unanswered question is how the stability of the open complex affects its ability to initiate RNA synthesis upon binding of NTPs. Here we address this question by comparing lifetimes, structural features, and patterns of short and long RNA synthesized from λPR promoter with either the λPR or T7A1 discriminator, using nitrocellulose filter binding assays, permanganate footprinting and transcription assays. We also compare lifetimes and structural features of λPR , T7A1 and ribosomal rrnBP1 promoters with either λPR or T7A1 discriminator, as well as examine abortive and productive transcription products by acrylamide gel and novel mass spectrometric analysis to determine the extent to which the discriminator region is responsible for open complex lifetime, and to elucidate its effects on transcription initiation. This work is supported through funding by the NIH (GM103061).

Probing DNA Binding, DNA Opening and Assembly of a Downstream Clamp During Transcription Initiation by E. coli RNA Polymerase at the λPR Promoter

Interactions between RNA polymerase and specific promoter DNA sequences trigger a precise progression of conformational changes in both biomolecules. These steps constitute the mechanism of DNA opening and the start of transcription; each step provides a critical checkpoint for regulatory input. Studies of E. coli RNA polymerase demonstrate that binding free energy alone opens the initiation bubble (−11 to +2), placing the +1 template base in the active site of the enzyme. Subsequent conformational changes are required to form the stable open complex RPo. Here we present recent fast footprinting experiments (hydroxyl radical, permanganate) that characterize the protection of the DNA backbone protection and the degree of thymine base unstacking in the three kinetically significant intermediates (I1, I2, I3) on the time scale of their formation. Experiments performed after mixing RNA polymerase and the λPR promoter DNA demonstrate that the DNA duplex containing the start site first loads in the RNA polymerase active site channel in duplex form to form I1, and that the subsequent step cooperatively opens the entire transcription bubble. In the back direction, [salt] upshift experiments demonstrate the existence of two unstable open intermediates, I2 and I3. Comparison of the degree of protection of the backbone in I2, I3 and RPo reveals differences between the nontemplate (NT) strand in these complexes but not the template (T) strand. We propose that the T strand is loaded first, followed by steps that reposition the NT strand, assemble the downstream clamp/jaw and thereby stabilize RPo. Experiments designed to test the role of the downstream RNA polymerase “jaw” and downstream DNA in the late steps will also be presented. This work was funded by NIH grant GM23467.