title: Observing Single RNA Polymerase Molecules Down to Base-Pair Resolution.
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.
authors.affiliation: Department of Biology, Stanford University, Stanford, CA, USA.
authors.forename: Anirban
authors.initials: A
authors.lastname: Chakraborty
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA, USA.
authors.forename: Cong A
authors.initials: CA
authors.lastname: Meng
authors.affiliation: Department of Biology and Department of Applied Physics, Stanford University, Stanford, CA, USA. sblock@stanford.edu.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Block
mesh_terms: D020029:Base Pairing; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008853:Microscopy / Q000379:methods; D052898:Optical Tweezers; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072760:Single Molecule Imaging / Q000379:methods*; D013057:Spectrum Analysis / Q000379:methods; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000737:chemistry / Q000378:metabolism
chemical_list: D035602:Transcriptional Elongation Factors; D012313:RNA; D012321:DNA-Directed RNA Polymerases
keywords: Optical trapping; RNA polymerase; RNA polymerase II; Single-molecule; Transcription