Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

Abstract

Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused-backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.

Keywords: RNA polymerase; RNAP; backtracking; pausing; transcription.

Fig. 1.

Single-round transcription quenched kinetics assay. (A) Representative promoter sequence used here (lacCONS promoter) to show how by changing the initially transcribed sequence (ITS; cyan), different NTP-starved states can be generated (RP_ITC=2, RP_ITC≤4,6,7, RD_E=11). Other regions of the promoter include the promoter recognition sequence (PRS; pink) and the elongation sequence (yellow), including a probe target complementary sequence (red). All promoters measured are described in Fig. S1. (B) Schematic of RNAP runoff transcription starting from a particular NTP-starved state (incubation with a partial set of NTPs for t_entrance). Upon supplementing all NTPs, transcription kinetics start and transcripts are quantified via hybridization to a ssDNA FRET probe for different incubation times (t_exit). (C) Example of quenched kinetics data generated from quantification of runoff transcripts. The example follows one repetition of the kinetics exiting from RP_ITC=2. (D) As an example for kinetic curve extraction, average runoff kinetics from various RP_ITC=2 are shown. The data points are averages of three repeats and the error bars are the SDs about these averages. The data are represented as points and the solid line represents the best-fit result to the model described in Methods. The best-fit values of the model parameters are shown in Table S1.

Fig. 2.

Quenched kinetics transcription results identify an initiation-related stalled state. Shown are runoff kinetics from various NTP-starved states. Kinetics starting from late initiation states (e.g., RP_ITC≤7, blue) are slower than from an earlier initiation state (e.g., RP_ITC=2, black). The data are represented as points and solid lines represent best-fit results to the model described in Methods. The best-fit values of the model parameters are shown in Table S1.

Fig. 3.

GreA suppresses the kinetic delay in transcription initiation. (A) Runoff transcription kinetics are slower when starting from RP_ITC≤7 (blue) than from RP_ITC=2 (black) (Fig. 2). (B) With 1 µM GreA, the delay in transcription initiation is reduced. The data are represented as points and solid lines represent best-fit results to the model described in Methods. The best-fit values of the model parameters are shown in Table S1. (C and D) Gel-based abortive initiation kinetics: Without GreA, NTP-starved RP_ITC≤7 produced abortive transcripts up to 7 bases long, whereas the 7-base product was not produced with 1 µM GreA, suggesting 2 bases of 3′-backtracked RNA were cleaved by RNAP in a GreA-catalyzed reaction during initiation. Band assignment is provided in Fig. S8A and the accompanying legend.

Fig. 4.

Backtracking in initiation correlates with RNAP pausing in the presence of equimolar NTPs. (A) Schematics of the magnetic tweezer transcription assay (Methods). (B and C) Representative bead extension trajectories shown for single-molecule transcription experiments without (B) or with (C) 1 µM GreA. Unwinding levels (gray lines) are shown, indicating different bubble sizes imposed by different RNAP states (below). Yellow lines highlight typical lifetimes in each state. (D–H) Unwinding levels and RP_ITC and RP_ITC* lifetimes of individual initiation events (i.e., averaging over all RP_ITC and RP_ITC* states seen from initiation to promoter escape) are summarized into unwinding–lifetime scatter plots without (D) or with (E) 1 µM GreA; their 1D projections are shown in F–H. Quadrant structure is built as discussed in Methods, Illustrations. Lifetime data in the absence or presence of GreA were first fitted to single or double exponentials based on goodness-of-fit. Then, the 2D data were temporally separated into events shorter than (cyan, absence of GreA; magenta, presence of GreA) or longer than (dark blue, absence of GreA; dark red, presence of GreA) the fast timescale for promoter escape (∼300 s) plus 1 SD. Similarly, the 2D data were spatially separated into events with apparent unwinding amplitude smaller or larger than the mean unwinding observed during short-duration escape events plus 1 SD. Apparent unwinding data associated with short- or long-escape timescales were then fitted to single- or double-Gaussian distributions based on goodness-of-fit and according to the color code described above. Dotted vertical lines are visual guides to the maximum of the respective Gaussian distributions. Twenty to fifty DNA templates, carrying the lacCONS promoter sequence, were used for each condition, with 5–10 transcription pulses per template.

Fig. 5.

A modified transcription initiation model. RNAP transcription initiation branches to (i) promoter clearance and transitions into elongation (black arrows) or into (ii) release of abortive transcripts (green and red arrows). After initial backtracking steps (e.g., from RP_ITC=7 to RP_ITC=7*), the complex can continue with either (iii) fast abortive transcript release (classic model, green arrow) or (iv) transition into a paused and backtracked state. Exit from the paused–backtracked state can occur either by successive slow backtracking steps (red arrow) or through intrinsic cleavage of RNA bases that are in the secondary channel, which prepares RNAP in, e.g., the RP_ITC=5 state. Upon cleavage, the complex can release the abortive transcript or reestablish RNA polymerization (e.g., from the RP_ITC=5 state).