Rate-limiting steps in RNA chain initiation.

Promoter-specific lags in the approach to the steady-state rate of abortive initiation were observed when Escherichia coli RNA polymerase was added to initiate the reaction. The lag times were related to the time required for free enzyme and free promoter to combine and isomerize into a functionally active complex. The lag times measured for several bacteriophage and bacterial promoters differed widely (10 sec to several minutes) and in most cases corresponded to the rate-limiting step in the initiation process. The unique advantage in using the abortive initiation reaction to measure the lags was that the binding and isomerization steps in a simple two-state model could be quantitated separately. The separation of the contributions of both steps was effected by deriving an equation to describe the rate of formation of the active binary complex. Results from experiments based on the theory showed a linear relationship between the observed lag times and the reciprocal enzyme concentration. The slope and intercept of the equation yielded quantitative estimates of the binding and isomerization steps in initiation. The analysis was applied to the bacteriophage T7 A2 and D promoters to show the bases for the differences in in vitro initiation frequency that have been observed for these promoters.