Discontinuous movement and conformational change during pausing and termination by T7 RNA polymerase

Abstract

Time-resolved characterization of T7 RNA polymerase pausing and terminating at a class II termination site has been carried out using site-specifically tethered chemical nucleases. The data indicate that T7RNAP normally moves uniformly down the template as a rigid body. However, at the class II site this movement is interrupted, and the leading edge of the polymerase moves further along the DNA than the trailing edge. This discontinuous movement may persist until it can no longer be accommodated by conformational changes in the elongation complex, at which point the polymerase can either pause or terminate. Termination, but not pausing, is abrogated by introduction of a disulfide bond between the polymerase fingers and thumb subdomains. The introduced cysteines disrupt a thumb-fingers salt-bridge and, under reducing conditions, this mutant enzyme shows reduced processivity coincident with extension of the RNA to 5 nt. These observations suggest that termination requires that the thumb and fingers subdomains move apart, in a reversal of a conformational change important for initially forming a stable transcription complex.