Kinetic measurements of Escherichia coli RNA polymerase association with bacteriophage T7 early promoters

Abstract

During infection of Escherichia coli by bacteriophage T7, E. coli RNA polymerase utilizes only three promoters (A1, A2, and A3). In vitro, the A promoters predominate at very low polymerase concentration, but at higher polymerase concentration the minor B, C, D, and E promoters are used with equal efficiency. The binding constant for the initial association of polymerase with promoters and the forward rate of isomerization to an "open" complex capable of initiation have been measured for the A1, A3, C, and D promoters using the abortive initiation reaction. At 80 mM KCl, 37 degrees C, both major and minor promoters isomerize rapidly (t1/2 = 10 to 30 s). In contrast, initial binding to the minor promoters (KI = 10(7) ) is at least 10-fold weaker than binding to major promoters KI greater than or equal to 10(8) ), suggesting promoter selectivity in the T7 system occurs at the point of initial binding. Association kinetics of the A1 and C promoters on intact T7 were the same as measured on restriction fragments of length greater than or equal to 500 base pairs. All open complexes dissociated with half-lives longer than 1 h. Overall equilibrium binding constants estimated from kinetic measurements ranged from 10(10) to greater than or equal to 10(11) M-1 for minor and major promoters, respectively. Data on heparin attack and abortive initiation turnover rates indicate open complex polymerase conformation may be different at the A1 and A3 promoters.