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

Joshua W. Shaevitz, Elio A. Abbondanzieri, Robert Landick, Steven M. Block
2003 Nature  
Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo 1 . 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 0 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
more » ... ssion 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 (,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. Recent studies have implicated the nucleolytic activity of RNA polymerase as part of a proofreading mechanism 2-4 , similar to that found in DNA polymerases 5 . A key feature of this proofreading mechanism is a short backtracking motion of the enzyme along the DNA template (directed upstream, opposite to the normal direction of transcriptional elongation). Similar rearward movements are thought to accompany the processes of transcriptional pausing 6-8 , arrest 9,10 , and transcription-coupled DNA repair 11 . During backtracking, the transcription bubble shifts and the DNA-RNA hybrid duplex remains in register, while the 3 0 end of the RNA transcript moves away from the active site, and may even protrude into the secondary channel (nucleotide entrance pore) of the enzyme 6,7,9 , blocking the arrival of ribonucleoside triphosphates (NTPs). In its backtracked state, RNAP is able to cleave off and discard the most recently added base(s) by endonucleolysis, generating a fresh 3 0 end at the active site for subsequent polymerization onto the nascent RNA chain. In this fashion, short RNA segments carrying misincorporated bases can be replaced, leading to the correction of transcriptional errors (Fig. 1a) . Accessory proteins have been identified that increase transcriptional fidelity by preferentially stimulating the cleavage of misincorporated nucleotides: GreA and GreB for E. coli RNA polymerase 4 and SII/TFIIS for eukaryotic RNA polymerase II 2,3 . We studied transcription by RNAP at physiological nucleotide concentrations using a new single-molecule assay together with improved optical trapping instrumentation. In combination, these achieve subnanometre resolution along with extremely low positional drift. Our current system is capable of near-base-pair resolution in individual records of RNAP displacements, and achieves base-pair resolution (,0.3 nm) in averages of multiple records. During an experiment, two beads are optically trapped in buffer above a microscope coverglass by independently steered laser traps. A recombinant derivative of E. coli RNAP is bound specifically via a biotin-avidin linkage to the smaller of two polystyrene beads, while the transcriptionally downstream end of the DNA template (or the upstream end, in the case of assisting forces) is bound to the larger bead via a digoxygenin-antibody linkage, forming a bead-RNAP-DNA-bead 'dumbbell' (Fig. 1b) . The tension in the DNA was kept nearly constant (8.4^0.8 pN), for loads both opposing and assisting transcription, by feedback control of the position of the optical trap holding the larger bead. A force of this magnitude has a negligible effect on transcription rates, and is well below the stall force for RNAP 12 . An opposing load was applied in all experiments, except where noted. Transcriptional elongation was observed by measuring the position of the smaller bead as the polymerase moved (Fig. 2a) . We chose to make the trap holding the larger bead an order of magnitude stiffer than that holding the smaller bead so that all motion appeared in the latter (see Methods). None of the components of the assay were attached to the coverglass surface: this isolates the system from drift of the microscope stage relative to the objective and other optics, which represented a major source of low-frequency noise in previous single-molecule studies 12-17 . Measured drift rates during our experiments were typically below 5 nm h 21 (data not shown). We recorded the transcriptional motion of over 150 individual RNAP molecules at 1 mM NTPs moving on a DNA template derived from the E. coli rpoB gene sequence. As previously noted 12, 13, 15, 17 , RNAP activity consists of periods of continuous motion interrupted by distinct pauses of variable duration (Fig. 2a) . The velocity during the continuous-motion phase averaged ,15 bp s 21 , but varied among molecules, consistent with earlier reports 12, 13, 15 . Computer analysis of RNAP records identified transcriptional pauses ranging from 1 s (our detection threshold) to more than 30 min. Only intervals where transcriptional elongation ceased and subsequently recovered were scored as pauses. Pausing events could 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. letters to nature NATURE | VOL 426 | 11 DECEMBER 2003 | www.nature.com/nature 684
doi:10.1038/nature02191 pmid:14634670 pmcid:PMC1483218 fatcat:cntorhl2n5cm3g3emvd773nvhy