Transcriptional pausing without backtracking

Abstract

T ranscriptional pausing by multisubunit RNA polymerases (RNAPs) plays key roles in gene regulation by coordinating RNAP movement with interactions of regulators and folding of the nascent RNA and, in metazoans, by helping program cycles of promoter-proximal transcription that poise RNAPII for gene expression (1, 2). However, mechanistic understanding of pausing is incomplete. Proposed mechanisms can be divided broadly into 2 classes ( Fig. 1): backtrack pausing, in which reverse translocation of RNAP dislodges the transcript 3Ј end from the active site and thereby prevents RNA synthesis; and (ii) nonbacktrack pausing, in which conformational rearrangements in the RNAP active site block the nucleotide addition cycle. Only a single example of nonbacktrack pausing, one for which the pause lifetime is increased by a nascent RNA hairpin, has been studied in biochemical detail (refs. 3 and 4 and references therein). Considerable disagreement exists over the contribution of nonbacktrack pauses to the ubiquitous pausing observed during both ensemble and single-molecule in vitro transcription experiments (5-9). An article in this issue of PNAS by Kireeva and Kashlev (10) now provides definitive evidence that, at least for bacterial RNAPs, significant nonbacktrack pausing occurs even without the contribution of pause RNA hairpins.Hairpin-stabilized pausing was initially studied for its role in synchronizing transcription with translation in the attenuation control regions of amino acid biosynthetic operons in enteric bacteria (e.g., the his and trp operons). Like all pauses studied to date, these sites cause kinetic partitioning of RNAPs between active and paused states (as opposed to affecting all transcribing RNAPs uniformly). These pause signals were found to be multipartite and to include synergistic contributions of different parts of RNAP's nucleic-acid scaffold both to the pause efficiency (the probability of entering the pause state at the kinetic branch between pausing and active elongation) and to pause lifetime (although the pause hairpin contributes only to pause lifetime). Detailed study of the his pause signal revealed that (i) it halted RNAP largely in the pretranslocated register ( Fig. 1; i.e., not backtracked), (ii) the active site was primarily inhibited for catalysis rather than for translocation or NTP binding, and (iii) inhibition appeared to arise by blocking formation of a 3-helix bundle composed of the RNAP bridge helix and trigger loop (4). This 3-helix bundle makes contacts to the NTP substrate in the A site required for rapid catalysis (11).A few other pause signals also have been studied to a limited extent, including (i) those in the early-transcribed region of bacteriophage T7, some of which were found to be RNA hairpinindependent (12), (ii) the Escherichia coli ops pause at which RNAP can enter backtrack states (13), and (iii) the HIV-1 ϩ62 pause (affecting human RNAPII), which appears to involve direct partitioning into a backtracked state (14). Single-molecule experiments with bacterial ...

A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure

“…Transcriptional pauses can be classified by their mechanisms based on studies of model bacterial RNAPs (see Box 1) [28-31]: ( i ) elemental pauses, at which incompletely understood conformational rearrangements of RNAP involving the clamp and bridge helix and mediated by interactions of the RNA-DNA scaffold inhibit nucleotide addition without backtracking, possibly by disfavoring the fully translocated register necessary for productive NTP binding (Figures 1B and 2) [29, 32-34]; ( ii ) backtrack pauses, in which RNA-DNA pairing energetics drive their reverse translocation through RNAP, removing the RNA 3′ nucleotide from the active site into the secondary channel (Figures 1C and 2) [28, 35, 36]; and ( iii ) hairpin-stabilized pauses, at which a nascent RNA structure invades the RNA exit channel, stabilizes an open-clamp conformation of the enzyme, and increases the pause dwell time (Figures 1D, 2, and 3) [28, 37]. Both backtrack and hairpin-stabilized pauses appear to form after the EC enters an initial elemental pause state.…”

Interactions between RNA polymerase and the “core recognition element” counteract pausing

“…Thus, each position of the consensus sequence is predicted to favor the pre-translocated state over the post-translocated state (−10 through effects on duplex stability, −1 through effects on active-center binding, and +1 through both). Accordingly, each position of the consensus PE would be predicted to increase the opportunity for the TEC to enter an “elemental pause” state (a state that, according to one view, is accessed from the pre-translocated state and serves as an obligatory intermediate for pausing; 7,8,10-11,20) or a “backtracked” state (a state that, according to another view, is accessed from the pre-translocated state and serves as the primary state for pausing; 2,16). …”

Discontinuous Galerkin Calculations for a Nonlinear PDE Model of DNA Transcription with Short, Transient and Frequent Pausing

“…It is known from empirical observation that during the transcription process, a polymerase frequently pauses along the DNA strand causing a transcriptional delay and affecting the instantaneous transcription speed, see [6,12,13,17]. One goal of the current research is to use numerical simulations of the PDE model described in Section 3 (with various selections of pause data) in order to quantify the effect of pauses and subsequent delays on the overall transcription rate of the ribosomal RNA.…”

“…Single molecule observation using optical traps indicate that the elongating polymerase transcribes rapidly but frequently pauses, and the duration of the pauses is roughly bi-modal [17] with means 1.2 ± 0.1 sec with amplitude 60% and 6 ± 0.4 sec with amplitude 40%. There are two types of transcriptional pauses of RNAP [13]. One type is referred to as backtracking pauses, and the other is called non-backtracking pauses, described in [11].…”