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Direct Observation of Abortive Initiation and Promoter Escape within Single Immobilized Transcription Complexes

Emmanuel Margeat ^*, Achillefs N. Kapanidis ^*, Philip Tinnefeld ^*, You Wang ^*, Jayanta Mukhopadhyay , Richard H. Ebright  and Shimon Weiss ^*

^* Department of Chemistry and Biochemistry, Department of Physiology, and the California NanoSystems Institute, University of California at Los Angeles, Los Angeles, California 90095; and Howard Hughes Medical Institute, Waksman Institute, and Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854

Correspondence: Address reprint requests to Emmanuel Margeat, E-mail: margeat{at}cbs.cnrs.fr or Shimon Weiss, E-mail: sweiss{at}chem.ucla.edu.

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.

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