title: Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes.
abstract: Using total-internal-reflection fluorescence microscopy equipped with alternating-laser excitation, we were able to detect abortive initiation and promoter escape within single immobilized transcription complexes. Our approach uses fluorescence resonance energy transfer to monitor distances between a fluorescent probe incorporated in RNA polymerase (RNAP) and a fluorescent probe incorporated in DNA. We observe small, but reproducible and abortive-product-length-dependent, decreases in distance between the RNAP leading edge and DNA downstream of RNAP upon abortive initiation, and we observe large decreases in distance upon promoter escape. Inspection of population distributions and single-molecule time traces for abortive initiation indicates that, at a consensus promoter, at saturating ribonucleoside triphosphate concentrations, abortive-product release is rate-limiting (i.e., abortive-product synthesis and RNAP-active-center forward translocation are fast, whereas abortive-product dissociation and RNAP-active-center reverse translocation are slow). The results obtained using this new methodology confirm and extend those obtained from diffusing single molecules, and pave the way for real-time, single-molecule observations of the transitions between various states of the transcription complex throughout transcription.
authors.affiliation: Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90095, USA.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Margeat
authors.forename: Achillefs N
authors.initials: AN
authors.lastname: Kapanidis
authors.forename: Philip
authors.initials: P
authors.lastname: Tinnefeld
authors.forename: You
authors.initials: Y
authors.lastname: Wang
authors.forename: Jayanta
authors.initials: J
authors.lastname: Mukhopadhyay
authors.forename: Richard H
authors.initials: RH
authors.lastname: Ebright
authors.forename: Shimon
authors.initials: S
authors.lastname: Weiss
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics / Q000502:physiology*; D024363:Transcription Initiation Site / Q000502:physiology*; D014158:Transcription, Genetic
chemical_list: D004247:DNA; D012321:DNA-Directed RNA Polymerases