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

Abstract

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 (ϳ1200 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. Transcription initiation by RNAP1 is an important regulatory step for gene expression in vivo. However, the details of transcription initiation are difficult to elucidate using conventional solution methods because (i) initiation consists of a series of transient intermediate steps between promoter binding and elongation, and (ii) within a population of actively transcribing RNAP molecules, only a small fraction is engaged in initiation at any given time. Most biochemical studies of transcription initiation utilize solution conditions which allow only a single enzymatic turnover, e.g. RNAP halted at a known position in the DNA sequence by ribonucleotide starvation or rapid mixing/quenching of the transcription assay. An attempt has been made to synchronize a population of T7 RNAP molecules in solution (1); however, synchrony is rapidly lost as the transitions between states are probabilistic events. The challenges associated with synchronizing a population of molecules can be avoided by making measurements on a single molecule. Previous single-molecule studies of transcription by Escherichia coli RNAP have only allowed DNA binding (2), elongation (artificially halted by ribonucleotide starvation) (3-8), and termination (9) to be observed in isolation from the other transcriptional states. No previous single-molecule transcription assay has allowed promoter recognition and the transition from initiation to elongation to be observed.T7 RNAP is a common paradigm for studies of transcription initiation, as it is a single-subunit enzyme sharing many of the biochemical characteristics of the more complex multi-subunit RNAPs from prokary...

Single-Molecule Studies of RNA Polymerase: Motoring Along

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Quasi-cellular systems: stochastic simulation analysis at nanoscale range

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A mutation in T7 RNA polymerase that facilitates promoter clearance

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