Transcription reinitiation properties of bacteriophage T7 RNA polymerase

Abstract

We have analyzed the kinetics of transcription initiation and reinitiation in vitro by one of the simplest and best characterized transcription machineries, bacteriophage T7 RNA polymerase (T7 RNAP). We used a short transcription unit with T7-specific promoter and terminator elements as a template, and a heparin challenge assay to distinguish the first transcription cycle from the subsequent ones. When present at sub-saturating concentrations with respect to template DNA, T7 RNAP could find its promoter and initiate the first transcription cycle in less than 1 min. Reinitiation under the same conditions proceeded more slowly, with only three new transcription cycles being completed in 10 min; after that time, reinitiation practically ceased. When the polymerase was in large excess over template DNA, however, reinitiation proceeded linearly for longer times, at a rate of 1 cycle/min. Our data suggest that polymerase recycling represents a critical step in T7 RNAP transcription, and that such a step may become rate-limiting for transcription at sub-saturating polymerase concentrations.