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


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.


  1. Erie, D. A., Yager, T. D. & von Hippel, P. H. The single-nucleotide addition cycle in transcription: a biophysical and biochemical perspective. Annu. Rev. Biophys. Biomol. Struct. 21, 379–415 (1992)

    CAS  Article  Google Scholar 

  2. Jeon, C. & Agarwal, K. Fidelity of RNA polymerase II transcription controlled by elongation factor TFIIS. Proc. Natl Acad. Sci. USA 93, 13677–13682 (1996)

    ADS  CAS  Article  Google Scholar 

  3. Thomas, M. J., Platas, A. A. & Hawley, D. K. Transcriptional fidelity and proofreading by RNA polymerase II. Cell 93, 627–637 (1998)

    CAS  Article  Google Scholar 

  4. Erie, D. A., Hajiseyedjavadi, O., Young, M. C. & von Hippel, P. H. Multiple RNA polymerase conformations and GreA: control of the fidelity of transcription. Science 262, 867–873 (1993)

    ADS  CAS  Article  Google Scholar 

  5. Kunkel, T. A. & Bebenek, K. DNA replication fidelity. Annu. Rev. Biochem. 69, 497–529 (2000)

    CAS  Article  Google Scholar 

  6. Komissarova, N. & Kashlev, M. RNA polymerase switches between inactivated and activated states by translocating back and forth along the DNA and the RNA. J. Biol. Chem. 272, 15329–15338 (1997)

    CAS  Article  Google Scholar 

  7. Nudler, E., Mustaev, A., Lukhtanov, E. & Goldfarb, A. The RNA-DNA hybrid maintains the register of transcription by preventing backtracking of RNA polymerase. Cell 89, 33–41 (1997)

    CAS  Article  Google Scholar 

  8. Marr, M. T. & Roberts, J. W. Function of transcription cleavage factors GreA and GreB at a regulatory pause site. Mol. Cell 6, 1275–1285 (2000)

    CAS  Article  Google Scholar 

  9. Komissarova, N. & Kashlev, M. Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3′ end of the RNA intact and extruded. Proc. Natl Acad. Sci. USA 94, 1755–1760 (1997)

    ADS  CAS  Article  Google Scholar 

  10. Reeder, T. C. & Hawley, D. K. Promoter proximal sequences modulate RNA polymerase II elongation by a novel mechanism. Cell 87, 767–777 (1996)

    CAS  Article  Google Scholar 

  11. Tornaletti, S., Reines, D. & Hanawalt, P. C. Structural characterization of RNA polymerase II complexes arrested by a cyclobutane pyrimidine dimer in the transcribed strand of template DNA. J. Biol. Chem. 274, 24124–24130 (1999)

    CAS  Article  Google Scholar 

  12. Neuman, K. C., Abbondanzieri, E. A., Landick, R., Gelles, J. & Block, S. M. Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking. Cell 115, 437–447 (2003)

    CAS  Article  Google Scholar 

  13. Forde, N. R., Izhaky, D., Woodcock, G. R., Wuite, G. J. & Bustamante, C. Using mechanical force to probe the mechanism of pausing and arrest during continuous elongation by Escherichia coli RNA polymerase. Proc. Natl Acad. Sci. USA 99, 11682–11687 (2002)

    ADS  CAS  Article  Google Scholar 

  14. Schafer, D. A., Gelles, J., Sheetz, M. P. & Landick, R. Transcription by single molecules of RNA polymerase observed by light microscopy. Nature 352, 444–448 (1991)

    ADS  CAS  Article  Google Scholar 

  15. Wang, M. D. et al. Force and velocity measured for single molecules of RNA polymerase. Science 282, 902–907 (1998)

    ADS  CAS  Article  Google Scholar 

  16. Yin, H., Landick, R. & Gelles, J. Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule. Biophys. J. 67, 2468–2478 (1994)

    ADS  CAS  Article  Google Scholar 

  17. Adelman, K. et al. Single molecule analysis of RNA polymerase elongation reveals uniform kinetic behavior. Proc. Natl Acad. Sci. USA 99, 13538–13543 (2002)

    ADS  CAS  Article  Google Scholar 

  18. Veigel, C. et al. The motor protein myosin-I produces its working stroke in two steps. Nature 398, 530–533 (1999)

    ADS  CAS  Article  Google Scholar 

  19. deCastro, M. J., Fondecave, R. M., Clarke, L. A., Schmidt, C. F. & Stewart, R. J. Working strokes by single molecules of the kinesin-related microtubule motor ncd. Nature Cell Biol. 2, 724–729 (2000)

    CAS  Article  Google Scholar 

  20. Nishiyama, M., Muto, E., Inoue, Y., Yanagida, T. & Higuchi, H. Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules. Nature Cell Biol. 3, 425–428 (2001)

    CAS  Article  Google Scholar 

  21. Aboul-ela, F., Koh, D., Tinoco, I. Jr & Martin, F. H. Base-base mismatches. Thermodynamics of double helix formation for dCA3XA3G + dCT3YT3G (X, Y = A,C,G,T). Nucleic Acids Res. 13, 4811–4824 (1985)

    CAS  Article  Google Scholar 

  22. Martin, F. H., Castro, M. M., Aboul-ela, F. & Tinoco, I. Jr Base pairing involving deoxyinosine: implications for probe design. Nucleic Acids Res. 13, 8927–8938 (1985)

    CAS  Article  Google Scholar 

  23. Borukhov, S., Sagitov, V. & Goldfarb, A. Transcript cleavage factors from E. coli. Cell 72, 459–466 (1993)

    CAS  Article  Google Scholar 

  24. Yildiz, A. et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003)

    ADS  CAS  Article  Google Scholar 

  25. Feng, G. H., Lee, D. N., Wang, D., Chan, C. L. & Landick, R. GreA-induced transcript cleavage in transcription complexes containing Escherichia coli RNA polymerase is controlled by multiple factors, including nascent transcript location and structure. J. Biol. Chem. 269, 22282–22294 (1994)

    CAS  PubMed  Google Scholar 

  26. Lang, M. J., Asbury, C. L., Shaevitz, J. W. & Block, S. M. An automated two-dimensional optical force clamp for single molecule studies. Biophys. J. 83, 491–501 (2002)

    ADS  CAS  Article  Google Scholar 

  27. Svoboda, K. & Block, S. M. Biological applications of optical forces. Annu. Rev. Biophys. Biomol. Struct. 23, 247–285 (1994)

    CAS  Article  Google Scholar 

  28. Wang, M. D., Yin, H., Landick, R., Gelles, J. & Block, S. M. Stretching DNA with optical tweezers. Biophys. J. 72, 1335–1346 (1997)

    ADS  CAS  Article  Google Scholar 

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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).

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