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J. Biol. Chem., Vol. 279, Issue 5, 3239-3244, January 30, 2004

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Promoter Binding, Initiation, and Elongation By Bacteriophage T7 RNA Polymerase

A SINGLE-MOLECULE VIEW OF THE TRANSCRIPTION CYCLE^*

Gary M. Skinner¶, Christoph G. Baumann¶, Diana M. Quinn, Justin E. Molloy||, and James G. Hoggett**

From the Department of Biology, University of York, York YO10 5YW, United Kingdom and ^||National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom

A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k_off= 2.9 s^–1) of T7 RNAP and promoter DNA, the transition to the elongation mode (k_for = 0.36 s^–1), and the processive synthesis (k_pol = 43 nt s^–1) and release of a gene-length RNA transcript (1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k_off > k_for), identifying a rate-limiting step between promoter DNA binding and promoter escape.

Received for publication, September 22, 2003

^* This research was supported by the Biotechnology and Biological Sciences Research Council (UK). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains the video.

Present address: Dept. of Physics, Univ. of Arizona, Tucson, AZ 85721.

^¶ These authors contributed equally to this work.

^** To whom correspondence should be addressed: Dept. of Biology (Area 10), P.O. Box 373, Univ. of York, York YO10 5YW, United Kingdom. Tel.: 44-1904-328670; Fax: 44-1904-328825; E-mail: jgh1{at}york.ac.uk.

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