Backtracking by single RNA polymerase molecules observed at near-base-pair resolution

Abstract

Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo. Its low error rate may be achieved by means of a 'proofreading' mechanism comprised of two sequential events. The first event (backtracking) involves a transcriptionally upstream motion of RNAP through several base pairs, which carries the 3' end of the nascent RNA transcript away from the enzyme active site. The second event (endonucleolytic cleavage) occurs after a variable delay and results in the scission and release of the most recently incorporated ribonucleotides, freeing up the active site. Here, by combining ultrastable optical trapping apparatus with a novel two-bead assay to monitor transcriptional elongation with near-base-pair precision, we observed backtracking and recovery by single molecules of RNAP. Backtracking events ( approximately 5 bp) occurred infrequently at locations throughout the DNA template and were associated with pauses lasting 20 s to >30 min. Inosine triphosphate increased the frequency of backtracking pauses, whereas the accessory proteins GreA and GreB, which stimulate the cleavage of nascent RNA, decreased the duration of such pauses.

Figure 1

RNA polymerase transcription and proofreading studied by optical trapping. a, During normal elongation, RNAP (green) moves forward (downstream) on the DNA (blue) as it elongates the nascent RNA (red). At each position along the template, RNAP may slide backward along the template, causing transcription to cease temporarily. From the backtracked state, polymerase can either slide forward again, returning to its earlier state (left) or cleave the nascent RNA (right) and resume transcriptional elongation. b, Cartoon of the experimental geometry employed for opposing force experiments (not to scale). Two beads (blue) are held in separate optical traps (red) in a force-clamp arrangement. The smaller bead (right) is bound to a single molecule of RNAP, while the larger bead (left) is bound to the downstream end of the DNA by non-covalent linkages (yellow). During transcriptional elongation, the beads are pulled together. Nearly all the motion appears as a displacement of the right bead (green arrow), which is held in a comparatively weaker trap.

Figure 2

Backtracking occurs upon entry into long, but not short, pauses. a, Transcription records of two individual RNAP molecules are shown, each moving over the same template sequence. Both traces contain multiple short pauses (most are too short to be seen on this timescale); one includes a very long pause (410 s, red trace). b, In some records of long pauses, backtracking could be seen by eye: a representative record is shown. The three phases of motion are indicated below the trace: phase 1 (backtracking, solid line), phase 2 (pause, dotted line), and phase 3 (recovery, solid line). c, A representative record of a short pause (3 s); such pauses do not exhibit backtracking. Data were recorded at 2 kHz and boxcar-filtered at 100 ms for display.

Figure 3

Averages of aligned long-pause records reveal details of backtracking and transcript cleavage events. a, Long-pause average of records at 1 mM NTPs (N = 56) displays a backtracking motion of ~5 bp (phase 1). Recovery (phase 3) is gradual, lasting ~5 s, before the resumption of normal elongation speed. b, Addition of ITP increases the frequency of long pauses that are indistinguishable from those in a (N = 26). c, Addition of GreA and GreB reduced the duration of long pauses. Recovery from these pauses (phase 3) was abrupt (N = 22), distinct from a and b. d, The short pause average (N = 56) displays no backtracking motion. Average records were smoothed with a 100-ms boxcar filter for display. ITP and the accessory proteins GreA and GreB affect both the frequency (e) and duration (f) of long pauses. See text.