Observing Single RNA Polymerase Molecules Down to Base-Pair Resolution

  • Anirban Chakraborty
  • Cong A. Meng
  • Steven M. Block
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1486)

Abstract

During transcriptional elongation, RNA polymerases (RNAP) employ a stepping mechanism to translocate along the DNA template while synthesizing RNA. Optical trapping assays permit the progress of single molecules of RNA polymerase to be monitored in real time, at resolutions down to the level of individual base pairs. Additionally, optical trapping assays permit the application of exquisitely controlled, external forces on RNAP. Responses to such forces can reveal details of the load-dependent kinetics of transcriptional elongation and pausing. Traditionally, the bacterial form of RNAP from E. coli has served as a model for the study of transcriptional elongation using optical traps. However, it is now feasible to perform optical trapping experiments using the eukaryotic polymerase, RNAPII, as well. In this report, we describe the methods to perform optical trapping transcriptional elongation assays with both prokaryotic RNAP and eukaryotic RNAPII. We provide detailed instructions on how to reconstitute transcription elongation complexes, derivatize beads used in the assays, and perform optical trapping measurements.

Key words

Single-molecule Optical trapping RNA polymerase RNA polymerase II Transcription 

Supplementary material

Capturing and testing a dumbbell with optical tweezers (WMV 14,606 kB).

References

  1. 1.
    Decker KB, Hinton DM (2013) Transcription regulation at the core: similarities among bacterial, archaeal, and eukaryotic RNA polymerases. Annu Rev Microbiol 67:113–139. doi:10.1146/annurev-micro-092412-155756 CrossRefGoogle Scholar
  2. 2.
    Hodges C, Bintu L, Lubkowska L, Kashlev M, Bustamante C (2009) Nucleosomal fluctuations govern the transcription dynamics of RNA polymerase II. Science 325(5940):626–628. doi:10.1126/science.1172926 CrossRefGoogle Scholar
  3. 3.
    Horn AE, Goodrich JA, Kugel JF (2014) Single molecule studies of RNA polymerase II transcription in vitro. Transcription 5(1):e27608CrossRefGoogle Scholar
  4. 4.
    Larson MH, Zhou J, Kaplan CD, Palangat M, Kornberg RD, Landick R, Block SM (2012) Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II. Proc Natl Acad Sci U S A 109(17):6555–6560. doi:10.1073/pnas.1200939109 CrossRefGoogle Scholar
  5. 5.
    Zhou J, Schweikhard V, Block SM (2013) Single-molecule studies of RNAPII elongation. Biochim Biophys Acta 1829(1):29–38. doi:10.1016/j.bbagrm.2012.08.006 CrossRefGoogle Scholar
  6. 6.
    Galburt EA, Grill SW, Wiedmann A, Lubkowska L, Choy J, Nogales E, Kashlev M, Bustamante C (2007) Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner. Nature 446(7137):820–823. doi:10.1038/nature05701 CrossRefGoogle Scholar
  7. 7.
    Galburt EA, Grill SW, Bustamante C (2009) Single molecule transcription elongation. Methods 48(4):323–332. doi:10.1016/j.ymeth.2009.04.021 CrossRefGoogle Scholar
  8. 8.
    Herbert KM, Greenleaf WJ, Block SM (2008) Single-molecule studies of RNA polymerase: motoring along. Annu Rev Biochem 77:149–176. doi:10.1146/annurev.biochem.77.073106.100741 CrossRefGoogle Scholar
  9. 9.
    Herbert KM, La Porta A, Wong BJ, Mooney RA, Neuman KC, Landick R, Block SM (2006) Sequence-resolved detection of pausing by single RNA polymerase molecules. Cell 125(6):1083–1094. doi:10.1016/j.cell.2006.04.032 CrossRefGoogle Scholar
  10. 10.
    Neuman KC, Abbondanzieri EA, Landick R, Gelles J, Block SM (2003) Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking. Cell 115(4):437–447CrossRefGoogle Scholar
  11. 11.
    Shaevitz JW, Abbondanzieri EA, Landick R, Block SM (2003) Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature 426(6967):684–687. doi:10.1038/nature02191 CrossRefGoogle Scholar
  12. 12.
    Abbondanzieri EA, Greenleaf WJ, Shaevitz JW, Landick R, Block SM (2005) Direct observation of base-pair stepping by RNA polymerase. Nature 438(7067):460–465. doi:10.1038/nature04268 CrossRefGoogle Scholar
  13. 13.
    Wang MD, Schnitzer MJ, Yin H, Landick R, Gelles J, Block SM (1998) Force and velocity measured for single molecules of RNA polymerase. Science 282(5390):902–907CrossRefGoogle Scholar
  14. 14.
    Yin H, Wang MD, Svoboda K, Landick R, Block SM, Gelles J (1995) Transcription against an applied force. Science 270(5242):1653–1657CrossRefGoogle Scholar
  15. 15.
    Neuman KC, Block SM (2004) Optical trapping. Rev Sci Instrum 75(9):2787–2809. doi:10.1063/1.1785844 CrossRefGoogle Scholar
  16. 16.
    Perkins TT (2014) Angstrom-precision optical traps and applications. Annu Rev Biophys 43:279–302. doi:10.1146/annurev-biophys-042910-155223 CrossRefGoogle Scholar
  17. 17.
    Schafer DA, Gelles J, Sheetz MP, Landick R (1991) Transcription by single molecules of RNA polymerase observed by light microscopy. Nature 352(6334):444–448. doi:10.1038/352444a0 CrossRefGoogle Scholar
  18. 18.
    Greenleaf WJ, Woodside MT, Abbondanzieri EA, Block SM (2005) Passive all-optical force clamp for high-resolution laser trapping. Phys Rev Lett 95(20):208102CrossRefGoogle Scholar
  19. 19.
    Visscher K, Gross SP, Block SM (1996) Construction of multiple-beam optical traps with nanometer-resolution position sensing. IEEE J Sel Top Quantum Electron 2(4):1066–1076. doi:10.1109/2944.577338 CrossRefGoogle Scholar
  20. 20.
    Fazal FM, Meng CA, Murakami K, Kornberg RD, Block SM (2015) Real-time observation of the initiation of RNA polymerase II transcription. Nature 525(7568):274–277. doi:10.1038/nature14882 CrossRefGoogle Scholar
  21. 21.
    Palangat M, Larson MH, Hu X, Gnatt A, Block SM, Landick R (2012) Efficient reconstitution of transcription elongation complexes for single-molecule studies of eukaryotic RNA polymerase II. Transcription 3(3):146–153. doi:10.4161/trns.20269 CrossRefGoogle Scholar
  22. 22.
    Komissarova N, Kireeva ML, Becker J, Sidorenkov I, Kashlev M (2003) Engineering of elongation complexes of bacterial and yeast RNA polymerases. Methods Enzymol 371:233–251. doi:10.1016/S0076-6879(03)71017-9 CrossRefGoogle Scholar
  23. 23.
    Kettenberger H, Armache KJ, Cramer P (2004) Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. Mol Cell 16(6):955–965. doi:10.1016/j.molcel.2004.11.040 CrossRefGoogle Scholar
  24. 24.
    Bustamante C, Marko JF, Siggia ED, Smith S (1994) Entropic elasticity of lambda-phage DNA. Science 265(5178):1599–1600CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Anirban Chakraborty
    • 1
  • Cong A. Meng
    • 2
  • Steven M. Block
    • 3
  1. 1.Department of BiologyStanford UniversityStanfordUSA
  2. 2.Department of ChemistryStanford UniversityStanfordUSA
  3. 3.Department of Biology and Department of Applied PhysicsStanford UniversityStanfordUSA

Personalised recommendations