Bacteriophage T7 E promoter: identification and measurement of kinetics of association with Escherichia coli RNA polymerase

Abstract

The initiation point for transcription from the Escherichia coli RNA polymerase E promoter on bacteriophage T7 has been determined to be at 36 835 base pairs (92.22 T7 units) from the left end of T7. The location was determined by RNA fingerprinting of a runoff transcription product. Kinetics of association for the E and the T7 A3 promoters were measured by using the abortive initiation assay for approach to steady-state turnover. The kinetic association constant, ka (=KBk2), for E was found to be over 10-fold slower than ka for A3. For the E promoter, ka = 1.2 X 10(6) M-1 s-1. For A3, we report ka greater than or equal to 4 X 10(7) M-1 s-1. This difference is due mostly to a 10-fold difference in the initial equilibrium constant, KB, for formation of the initial polymerase-promoter complex. The rate of isomerization, k2, of the initial complex to the open polymerase-promoter complex for the E promoter was only 2-fold slower than k2 for the A3 promoter. Various numerical methods for calculation of the kinetic parameters are discussed and compared. We argue that a nonlinear analysis provides the most reliable means of data analysis.