Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes.
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.
Margeat
Emmanuel
E
Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90095, USA.
Kapanidis
Achillefs N
AN
Tinnefeld
Philip
P
Wang
You
Y
Mukhopadhyay
Jayanta
J
Ebright
Richard H
RH
Weiss
Shimon
S
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
D004247:DNA; D012321:DNA-Directed RNA Polymerases