E. coli NusG inhibits backtracking and accelerates pause-free transcription by promoting forward translocation of RNA polymerase

Abstract

NusG is an essential transcription factor in Escherichia coli that is capable of increasing the overall rate of transcription. Transcript elongation by RNA polymerase (RNAP) is frequently interrupted by pauses of varying durations, and NusG is known to decrease the occupancy of at least some paused states. However, it has not been established whether NusG enhances transcription chiefly by (1) increasing the rate of elongation between pauses, (2) reducing the lifetimes of pauses, or (3) reducing the rate of entry into paused states. Here, we studied transcription by single molecules of RNAP under various conditions of ribonucleoside triphosphate concentration, applied load, and temperature, using an optical trapping assay capable of distinguishing pauses as brief as 1 s. We found that NusG increases the rate of elongation, that is, the pause-free velocity along the template. Because pauses are off-pathway states that compete with elongation, we observed a concomitant decrease in the rate of entry into short-lifetime, paused states. The effects on short pauses and elongation were comparatively modest, however. More dramatic was the effect of NusG on suppressing entry into long-lifetime ("stabilized") pauses. Because a significant fraction of the time required for the transcription of a typical gene may be occupied by long pauses, NusG is capable of exerting a significant modulatory effect on the rates of RNA synthesis. The observed properties of NusG were consistent with a unified model where the function of this accessory factor is to promote transcriptionally downstream motion of the enzyme along the DNA template, which has the effect of forward-biasing RNAP from the pre-translocated state toward the post-translocated state.

Figure 1. Single-molecule transcription assay and the effect of NusG

(a) A cartoon showing the experimental geometry for the single-molecule assay (not to scale). Two polystyrene beads (light blue) are each held in separate optical traps (pink) above a coverglass. A biotin label (black) on RNAP (green) was used to attach RNAP to the smaller bead via an avidin linkage (yellow). The 3′-downstream end of the DNA (dark blue), labeled with digoxigenin (orange), is bound to a larger bead by an anti-digoxigenin antibody (purple). Transcription proceeds in the direction indicated (green arrow) while RNAP experiences a hindering load. By appending the digoxigenin label instead to the 3′-upstream end of the DNA, the direction of the applied load relative to transcription can be reversed. (b) Representative records of transcription by single RNAP molecules along the ops repeat template vs. time, in the absence (red; 4 traces shown of 57) or presence of 2 μM NusG (blue; 4 traces shown of 19), shown after computer alignment. Records were obtained at ~27 °C with 1 mM NTPs under a 5 pN hindering load. The positions of ops pause sequences (horizontal grey lines) and the rrnB T1 terminator (horizontal yellow line) are indicated. Note that the blue records proceed more quickly and display fewer long pauses.

Figure 2. Effect of NusG on long pauses

(a) Long pause density (pauses/kb) (mean ± bootstrapped std. dev.) in the presence (blue) or absence (red) of NusG under the conditions indicated below (b). (b) Long pause lifetime (mean ± bootstrapped std. dev.) in the presence (blue) or absence (red) of NusG under the conditions indicated.

Figure 3. Ensemble distributions of instantaneous velocity

Histogram distributions of the instantaneous velocity computed from records of individual transcribing RNAP molecules in the absence (red) or presence (blue) of 2 μM NusG were combined, a procedure that gives equal statistical weight to all positions on the template. Experimental assay conditions are shown on the right. The distributions were well fit by Gaussians (solid black lines), with the central value providing an estimate of the mean pause-free elongation velocity (mean values and std. errs. of fits are shown inset).

Figure 4. A simplified transcription pathway and the effect of NusG on short pauses

(a) Kinetics of a simplified model for elongation involving one off-pathway pause state. Forward elongation is depicted by a single transition occurring at a rate k_n. Pause efficiency, PE, is given by the ratio of the rate for pausing, k_p, to the rate of either pausing or elongating, (k_p+ k_n). The pause lifetime, L, is given by the reciprocal of the rate for escaping the pause, k_−p (b) The density of short pauses was calculated for each single-molecule record by tallying the number of pauses below 20 s and dividing by the distance transcribed, measured in kilobase pairs. The mean density ± std. error is shown for the assay conditions indicated. (c) The mean duration of short-lifetime pauses. The mean time ± std. error is shown for the assay conditions indicated.

Figure 5. Sequence-resolved effects of NusG on repeating templates

(a) Engineered transcription templates used here and in ref. . Templates carry a sequence of eight repeat motifs (pink) that begins ~1100 bp beyond a T7 A1 promoter (dark green), from which transcription was initiated, and ends ~80 bp before an rrnB T1 terminator (yellow). Templates were labeled with digoxigenin on the transcriptionally upstream end (for assisting loads; solid orange) or the transcriptionally downstream end (for hindering loads; transparent orange). Repeat motifs (red arrow) consist of leader sequence (pink) together with an ops pause sequence (gray), flanked by sequence elements from the rpoB gene (light green) and DNA derived from sites used in cloning (blue). (b) Average log dwell-time histogram of aligned data (acquired at 1 mM ATP, CTP, and UTP, plus 250 μM GTP, ~23 °C under 7.5 pN assisting load) computed from all repeats of the motif in the absence (red; N = 16 molecules, 81 records) or in the presence of NusG (blue; N = 14 molecules, 81 records), plotted along with bootstrapped standard deviations (white zones). The major pause sites are labeled. The table below shows pause statistics computed for the three most prominent pause sites in the repeat (a, d, and ops) in the presence or absence of NusG. (c) Average log dwell-time histogram for aligned data (acquired at 1 mM NTPs, ~27 °C, and 5 pN hindering load) computed from all 8 repeats of the ops repeat motif in the absence of NusG (red; N = 36 molecules, 175 records) and in the presence of NusG (blue; N = 13 molecules, 49 records), plotted with the bootstrapped standard deviations (white zones). The table below shows pause statistics computed for the three most prominent pause sites in the repeat (a, d, and ops) in the presence or absence of NusG.

Figure 6. Effect of NusG on a transcription run-off assay for RNA

Transcription of RNA was initiated by mixing 25 nM of a short template carrying a single copy of the repeat motif region, 40 nM biotin-tagged RNAP, and 10 mM ATP, GTP, and ³²P-CTP and incubated for 15 min at 37°C. If present, 100 nM NusG was added to halted complexes and incubated for 3 min at 37°C. Halted complexes were equilibrated to room temperature and elongation was restarted by adjustment to 1 mM ATP, CTP, and UTP, and 250 μM GTP with 100 mg/ml rifampicin. Samples were removed at 5, 15, 30, 60, and 120 s intervals. The marker lane (M) is fX714 DNA digested with HinfI; sizes of fragments (in basepairs) are indicated on the right, pauses are identified on the left.

Figure 7. A unified pathway for elongation and pausing

The main pathway for transcript elongation is shown (light blue boxes; top row; adapted from ref. 38). In a Brownian ratchet mechanism, RNAP oscillates stochastically between pre- and post-translocated states prior to the reversible binding of NTP followed by the (nearly) irreversible condensation reaction and pyrophosphate release, which rectify this motion in the transcriptionally downstream direction. The displacement associated with translocation, δ, corresponds to the longitudinal distance subtended by a single base pair. The elemental pause state is depicted (middle row, orange box; adapted from ref 14), shown branching from the pre-translocated state: entry into this state does not involve translocation. The long-lifetime, backtracked pause state (bottom row; orange box) is entered via the elemental pause state, and involves the upstream translocation of RNAP through one or more base pairs, Nδ. Our modeling suggests that the addition of NusG promotes the downstream motion of RNAP, affecting those transitions that involve translocation (red arrows).