Ga. Diaz et al., THE STABILITY OF ABORTIVELY CYCLING T7 RNA-POLYMERASE COMPLEXES DEPENDS UPON TEMPLATE CONFORMATION, Biochemistry, 35(33), 1996, pp. 10837-10843
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.