Single-pass transcription by T7 RNA polymerase

Abstract

RNA molecules can be conveniently synthesized in vitro by the T7 RNA polymerase (T7 RNAP). In some experiments, such as co-transcriptional biochemical analyses, continuous synthesis of RNA is not desired. Here, we propose a method for a single-pass transcription that yields a single transcript per template DNA molecule using the T7 RNAP system. We hypothesized that stalling the polymerase downstream of the promoter region and subsequent cleavage of the promoter by a restriction enzyme (to prevent promoter binding by another polymerase) would allow synchronized production of a single transcript per template. The single-pass transcription was verified in two different scenarios: a short self-cleaving ribozyme and a long mRNA. The results show that a controlled single-pass transcription using T7 RNAP allows precise measurement of co-transcriptional ribozyme activity, and this approach will facilitate the study of other kinetic events.