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

Abstract

Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo1. 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 (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.

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Figure 1: RNA polymerase transcription and proofreading studied by optical trapping.
Figure 2: Backtracking occurs upon entry into long, but not short, pauses.
Figure 3: Averages of aligned long-pause records reveal details of backtracking and transcript cleavage events.

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Acknowledgements

We acknowledge intellectual contributions from J. Gelles, and we thank the entire Block Laboratory, especially K. Neuman, for support and discussions. We also thank A. Meyer for reading of the original manuscript. This work was supported by grants from the NIGMS.

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Correspondence to Steven M. Block.

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Shaevitz, J., Abbondanzieri, E., Landick, R. et al. Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature 426, 684–687 (2003). https://doi.org/10.1038/nature02191

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