THE STABILITY OF ABORTIVELY CYCLING T7 RNA-POLYMERASE COMPLEXES DEPENDS UPON TEMPLATE CONFORMATION

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 upo n the conformation of the promoter, and that the degree to which the t emplate 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 (t(1/2) < 1 min). However, on a supercoiled template, the IC has a stability that is comparable to that of a paused elongation c omplex (t(1/2) = 14 min). At a synthetic promoter that is single stran ded in the initiation region (from -5 and downstream), the polymerase forms a highly stable complex (t(1/2) > 30 min) even in the absence of RNA synthesis. These findings are important to our understanding of t he transition from the IC to an EC.