Using Solutes and Kinetics to Probe Large Conformational Changes in the Steps of Transcription Initiation

Ruff, Emily F.; Kontur, Wayne S.; Record, M. Thomas

doi:10.1007/978-1-4939-2392-2_14

Cited by 4 publications

(7 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For filterbinding assays and permanganate footprinting experiments, HTOP or HBOT was kinase-labeled using T4 polynucleotide kinase and γ-[ Nitrocellulose Filter-Binding Dissociation Assays. Dissociation kinetic assays were performed as previously described (29,33). RNAP-promoter OCs were formed by incubating 2-6 nM RNAP for 1 h in binding buffer (BB) (see SI Appendix, General Procedures for composition) at 37°C with γ-32 P-radiolabeled promoter DNA (∼36,000 cpm).…”

Section: Methodsmentioning

confidence: 99%

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

Henderson

Felth

Molzahn

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

128

View full text Add to dashboard Cite

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...

show abstract

Section: Methodsmentioning

confidence: 99%

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

Henderson

Felth

Molzahn

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

128

View full text Add to dashboard Cite

show abstract

“…Filter binding assays were used to determine the amount of operator DNA retained on a nitrocellulose filter membrane in a complex with lac repressor protein ( 30 , 32 ). All thermodynamic and kinetic assays were performed in BB.…”

Section: Methodsmentioning

confidence: 99%

“…This research served as proof-of-principle of the use of solutes as probes of changes in surface area in biopolymer processes. Solute and salt effects on the thermodynamics and kinetics of RNA polymerase–promoter DNA open complex formation and stabilization were also used to identify and characterize large conformational changes and new interfaces in the key intermediates and in the DNA opening-closing TS in transcription initiation ( 30 – 32 ).…”

Section: Introductionmentioning

confidence: 99%

The mechanism and high-free-energy transition state of lac repressor–lac operator interaction

Sengupta

Michael

Shkel

et al. 2017

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

Significant, otherwise-unavailable information about mechanisms and transition states (TS) of protein folding and binding is obtained from solute effects on rate constants. Here we characterize TS for lac repressor(R)–lac operator(O) binding by analyzing effects of RO-stabilizing and RO-destabilizing solutes on association (ka) and dissociation (kd) rate constants. RO-destabilizing solutes (urea, KCl) reduce ka comparably (urea) or more than (KCl) they increase kd, demonstrating that they destabilize TS relative to reactants and RO, and that TS exhibits most of the Coulombic interactions between R and O. Strikingly, three solutes which stabilize RO by favoring burial/dehydration of amide oxygens and anionic phosphate oxygens all reduce kd without affecting ka significantly. The lack of stabilization of TS by these solutes indicates that O phosphates remain hydrated in TS and that TS preferentially buries aromatic carbons and amide nitrogens while leaving amide oxygens exposed. In our proposed mechanism, DNA-binding-domains (DBD) of R insert in major grooves of O pre-TS, forming most Coulombic interactions of RO and burying aromatic carbons. Nucleation of hinge helices creates TS, burying sidechain amide nitrogens. Post-TS, hinge helices assemble and the DBD-hinge helix-O-DNA module docks on core repressor, partially dehydrating phosphate oxygens and tightening all interfaces to form RO.

show abstract

“…Dissociation kinetic assays were performed as previously described [7, 47]. OCs were formed by incubating 2-5 nM RNAP (total concentration) with ~0.5 nM 32 P-labeled promoter DNA fragment for 0.5-3 hours at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

“… a Previous determinations of k d for WT RNAP-λP R OC: k d = (1.9 ± 0.6) × 10 −5 s −1 (DNA fragment extending from −115 to +76 was cut out of a plasmid and labelled) [5]; k d = (2.2 ± 0.3) × 10 −5 s −1 (890 bp fragment containing both λP R and λP RM was cut out of a plasmid and labelled) [47]. …”

Section: Highlightsmentioning

confidence: 99%

E. coli RNA Polymerase Determinants of Open Complex Lifetime and Structure

Ruff

Drennan

Michael

et al. 2015

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Using Solutes and Kinetics to Probe Large Conformational Changes in the Steps of Transcription Initiation

Cited by 4 publications

References 24 publications

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

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

The mechanism and high-free-energy transition state of lac repressor–lac operator interaction

E. coli RNA Polymerase Determinants of Open Complex Lifetime and Structure

Contact Info

Product

Resources

About