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

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Abstract
The initiation point for transcription from the E. coli RNA polymerase E promoter on bacteriophage T7 was determined to be at 36,835 base pairs (92.22 T7 U) 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 .times. 106 M-1 s-1. For A3, ka .gtoreq. 4 .times. 107 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. A nonlinear analysis provides the most reliable means of data analysis.