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

Abstract

A single-molecule transcription assay has been devel- oped that allows, for the first time, the direct observa- tion 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 es- cape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template re- lease, transcription re-initiation on the same DNA tem- plate 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 (koff  2.9 s1) of T7 RNAP and promoter DNA, the tran- sition to the elongation mode (kfor  0.36 s1), and the processive synthesis (kpol  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 (koff > kfor), identifying a rate-limiting step be- tween promoter DNA binding and promoter escape.