Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation by<i>E. coli</i>RNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex

Sreenivasan, Raashi; Shkel, Irina A.; Chhabra, Munish; Drennan, Amanda C.; Heitkamp, Sara; Wang, H.-C.; Sridevi, M. A.; Plaskon, Dylan; McNerney, Christina; Callies, Katelyn; Cimperman, Clare Kai; Record, M. Thomas

doi:10.1101/2020.02.04.932780

Cited by 5 publications

(10 citation statements)

References 72 publications

(43 reference statements)

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“…The thermodynamic, kinetic, and footprinting information available for these intermediates support the proposal that IC is I 3 . I 2 is unstable with respect to I 3 and/or RP O at higher temperatures and unstable with respect to CCs at lower temperatures (11,17,26,27). Conversion of I 2 to I 3 involves the folding of 100 to 150 amino acid residues of RNAP DME (27) and is thought to strengthen contacts between the proximal downstream duplex, the β lobe, and the β' clamp (28).…”

Section: Evidence That Only the I 3 Intermediate Oc And Not Rp O Or I...mentioning

confidence: 99%

“…To understand regulation, the detailed mechanisms of these stages must be understood. Recent structural (5)(6)(7)(8)(9), kinetic-mechanistic (10)(11)(12)(13)(14)(15)(16)(17)(18)(19), and high-throughput sequencing studies (20,21) have greatly advanced our understanding of initiation, but much remains to be learned for the rational discovery of drug targets and for the design of synthetic promoters optimized for specific applications in molecular biology and medical biotechnology (22).…”

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confidence: 99%

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Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise

Plaskon

Henderson

Felth

et al. 2021

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

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Transcription initiation is highly regulated by promoter sequence, transcription factors, and ligands. All known transcription inhibitors, an important class of antibiotics, act in initiation. To understand regulation and inhibition, the biophysical mechanisms of formation and stabilization of the “open” promoter complex (OC), of synthesis of a short RNA–DNA hybrid upon nucleotide addition, and of escape of RNA polymerase (RNAP) from the promoter must be understood. We previously found that RNAP forms three different OC with λPR promoter DNA. The 37 °C RNAP-λPR OC (RPO) is very stable. At lower temperatures, RPO is less stable and in equilibrium with an intermediate OC (I3). Here, we report step-by-step rapid quench-flow kinetic data for initiation and growth of the RNA–DNA hybrid at 25 and 37 °C that yield rate constants for each step of productive nucleotide addition. Analyzed together, with previously published data at 19 °C, our results reveal that I3 and not RPO is the productive initiation complex at all temperatures. From the strong variations of rate constants and activation energies and entropies for individual steps of hybrid extension, we deduce that contacts of RNAP with the bubble strands are disrupted stepwise as the hybrid grows and translocates. Stepwise disruption of RNAP-strand contacts is accompanied by stepwise bubble collapse, base stacking, and duplex formation, as the hybrid extends to a 9-mer prior to disruption of upstream DNA–RNAP contacts and escape of RNAP from the promoter.

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Section: Evidence That Only the I 3 Intermediate Oc And Not Rp O Or I...mentioning

confidence: 99%

mentioning

confidence: 99%

Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise

Plaskon

Henderson

Felth

et al. 2021

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

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“…From an analysis of changes in Cy3-to-Cy5 FRET efficiency for probes located at −100 and +14 positions of promoter DNA in the steps of λP R OC formation, we previously deduced that the β’ clamp opens to admit the downstream duplex and then closes on the duplex in the most advanced CC (designed I 1L ) [6]. We proposed that clamp opening accompanies opening the downstream duplex to form the unstable I 2 open intermediate from I 1L .…”

Section: Discussionmentioning

confidence: 99%

“…At the λ P R promoter, as summarized in Fig. 1 (see [5] and [6]), kinetic-mechanistic evidence indicates that initial binding triggers a series of conformational changes beginning with bending and wrapping of far-upstream duplex DNA (the UP element and beyond) on RNAP to contact the β’ clamp and form an ensemble of intermediate closed complexes collectively designated as I 1 . In the most advanced I 1 species, the downstream promoter duplex is bent into the RNAP cleft and the β’ clamp is closed.…”

Section: Introductionmentioning

confidence: 99%

Roles of Clamp Closing and Allosteric Effects of Discriminator and Upstream Interactions on Downstream Elements in Stabilizing an E. coli RNA Polymerase-Promoter Open Complex

Wang

Stronek

Record

2022

Preprint

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E. coli RNA polymerase (α2ββ’ωσ70) forms stable open complexes (OC) at λPR and T7A1 promoters at 37 °C at similar rates but with very different lifetimes. To probe the origins of these differences, we determine OC lifetimes for full-length (FL) and downstream-truncated (DT) λPR and T7A1 promoter variants with eight combinations of discriminators and upstream elements. We find the discriminator is the major determinant of OC lifetime, while upstream elements modulate the discriminator effect. The very different lifetimes of these stable OC arise primarily from differences in , the free energy change for converting I2 (the initial open intermediate) to stable OC. is twice as favorable for λPR-discriminator variants as for T7A1-discriminator variants. Truncation at +6 (DT+6) eliminates most differences in , destabilizing λPR-discriminator variants without affecting T7A1-discriminator variants. All DT+6 OC are much more stable and longer-lived than I2. Urea greatly destabilizes OC, primarily affecting RNAP interactions with +6 and upstream DNA. From these findings we deduce that stabilization of I2 involves closing the β’-clamp with extensive coupled folding to contact the region of promoter DNA from the transcription start-site to +6. Lifetime differences between DT+6, DT+12 and FL promoters allow dissection of contributions to OC stability from interactions of regions of the downstream duplex with downstream mobile elements (DME; β-lobe, β’–clamp, β’-jaw). We propose an allosteric network by which differences in discriminator and −10 sequence are sensed by σ1.2 and β-gate loop and transmitted to σ1.1 and β-lobe to affect DME-duplex interactions that determine OC lifetime.

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“…In the initial specific closed complex (RPC), the start site region of duplex promoter DNA is not contacted by RNAP (10,15). Recent kinetic-mechanistic and structural research have increased our understanding of the mechanisms by which the start site region is opened by RNAP using binding free energy and the template strand is brought to the vicinity of the RNAP active site to form the initial (unstable) open complex (I2) (11,(15)(16)(17)(18)(19)(20)(21)(22).…”

Section: Introductionmentioning

confidence: 99%

Kinetic-Mechanistic Evidence for WhichE. coliRNA Polymerase-λP_ROpen Promoter Complex Initiates and for Stepwise Disruption of Contacts in Bubble Collapse

Plaskon

Henderson

Felth

et al. 2020

Preprint

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In transcription initiation, specific contacts between RNA polymerase (RNAP) and promoter DNA are disrupted as the RNA-DNA hybrid advances into the cleft, resulting in escape of RNAP. From the pattern of large and small rate constants for steps of initiation at λPR promoter at 19°C, we proposed that in-cleft interactions are disrupted in extending 3-mer to 5-mer RNA, −10 interactions are disrupted in extending 6-mer to 9-mer, and −35 interactions are disrupted in extending 10-mer to 11-mer, allowing RNAP to escape. Here we test this mechanism and determine enthalpic and entropic activation barriers of all steps from kinetic measurements at 25°C and 37°C. Initiation at 37°C differs significantly from expectations based on lower-temperature results. At low concentration of the second iNTP (UTP), synthesis of full-length RNA at 37°C is slower than at 25°C and no transient short RNA intermediates are observed, indicating a UTP-dependent bottleneck step early in the 37°C mechanism. Analysis reveals that the 37°C λPR OC (RPO) cannot initiate and must change conformation to a less-stable initiation complex (IC) capable of binding the iNTP. We find that IC is the primary λPR OC species below 25°C, and therefore conclude that IC must be the I3 intermediate in RPO formation. Surprisingly, Arrhenius activation energy barriers to five steps where RNAP-promoter in-cleft and −10 contacts are disrupted are much smaller than for other steps, including a negative barrier for the last of these steps. We interpret these striking effects as enthalpically-favorable, entropically-unfavorable, stepwise bubble collapse accompanying disruption of RNAP contacts.SignificanceTranscription initiation is highly regulated. To understand regulation, mechanisms of initiation and escape of RNA polymerase (RNAP) from the promoter must be understood. RNAP forms a highly-stable open complex (RPO) with λPR promoter at 37°C. From experiments determining effects of temperature on rate constants for each step of RNA synthesis, we find that RPO cannot bind the initiating nucleotides, that the I3 intermediate and not RPO is the initiation complex, and that contacts of RNAP with single-stranded DNA of the discriminator and −10 region and with −35 duplex DNA are disrupted stepwise as the RNA-DNA hybrid moves into the cleft. Evidence is obtained for stepwise bubble collapse and base stacking accompanying disruption of interactions of the single-stranded discriminator and −10 regions with RNAP.

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Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation byE. coliRNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex

Cited by 5 publications

References 72 publications

Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise

Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise

Roles of Clamp Closing and Allosteric Effects of Discriminator and Upstream Interactions on Downstream Elements in Stabilizing an E. coli RNA Polymerase-Promoter Open Complex

Kinetic-Mechanistic Evidence for WhichE. coliRNA Polymerase-λP_ROpen Promoter Complex Initiates and for Stepwise Disruption of Contacts in Bubble Collapse

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Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation byE. coliRNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex

Cited by 5 publications

References 72 publications

Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise

Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise

Roles of Clamp Closing and Allosteric Effects of Discriminator and Upstream Interactions on Downstream Elements in Stabilizing an E. coli RNA Polymerase-Promoter Open Complex

Kinetic-Mechanistic Evidence for WhichE. coliRNA Polymerase-λPROpen Promoter Complex Initiates and for Stepwise Disruption of Contacts in Bubble Collapse

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Kinetic-Mechanistic Evidence for WhichE. coliRNA Polymerase-λP_ROpen Promoter Complex Initiates and for Stepwise Disruption of Contacts in Bubble Collapse