Interaction of RNA Polymerase with Promoters from Bacteriophage fd

Abstract

Replicative form DNA of bacteriophage fd, which was fragmented with the restriction endonuclease II from Haemophilus parainfluenzae (endo R .cntdot. HpaII), was reacted with Escherichia coli RNA polymerase [EC 2.7.7.6]; the resulting stable preinitiation complexes were analyzed using the filter binding assay followed by gel electrophoresis. At 120 mM KCl the 1st-order rate constants for complex decay were 10-2-10-6 s-1. The 2nd-order rate constants for complex formation were .apprx. 106-107 M-1 s-1. From these values the calculated association constants for the individual promoters were 2 .times. 10-8-2 .times. 10-11 M-1. The rate of formation and the stability of promoter complexes was enhanced in superhelical DNA. No evidence was found for stable promoter-specific closed complexes consisting of enzyme and helical DNA. This and the kinetic data suggest that the unwinding of base pairs is already important early in promoter selection, and not only for the formation of the final open complex. The initiation of RNA synthesis from the preinitiation complex was faster than complex dissociation and complex formation for all promoters. Consequently, the initiation efficiency of a promoter is determined by the rate of complex formation, and not by its affinity for the enzyme. No correlation was found between the relative order of the fd promoters for the binding and the dissociation reaction. This is explained by different structural determinants for the 2 reactions, located in different parts of the promoter DNA.