Template strand switching by T7 RNA polymerase

Abstract

T7 RNA polymerase (RNAP) is able to traverse a variety of discontinuities in the template (T) strand of duplex DNA, including nicks, gaps, and branched junctions in which the 3' end of the T strand is not complementary to the non-template (NT) strand. The products represent a faithful copy of the T strand, with no insertions or deletions. On double- stranded templates having protruding 3' ends the polymerase is able to insert the free 3' end of the NT strand and to utilize this as a new T strand ('turn around transcription'), resulting in the anomalous production of high molecular weight transcripts. The capacity of T7 RNAP to bypass interruptions in the T strand depends upon the stability of the elongation complex. Sequences that are expected to stabilize a local RNA:DNA hybrid (such as the presence of a C6 tract in the T strand) dramatically reduce dissociation of the RNAP while still allowing the enzyme to insert a new 3' end. Similar effects on RNAP release are observed when the enzyme reaches the end of a template (i.e. when synthesizing runoff products), resulting in markedly different yields of RNA product during multiple rounds of transcription.