The stability of abortively cycling T7 RNA polymerase complexes depends upon template conformation.

Abstract

We have developed a promoter competition assay to determine whether T7 RNA polymerase dissociates from its template during abortive cycling. We find that the stability of the initiation complex (IC) depends upon the conformation of the promoter, and that the degree to which the template is unwound contributes importantly to the stability of the IC. On linear DNA or a relaxed plasmid template, the stability of the IC is very low (t1/2 < 1 min). However, on a supercoiled template, the IC has a stability that is comparable to that of a paused elongation complex (t1/2 = 14 min). At a synthetic promoter that is single stranded in the initiation region (from -5 and downstream), the polymerase forms a highly stable complex (t1/2 > 30 min) even in the absence of RNA synthesis. These findings are important to our understanding of the transition from the IC to an EC.