Interaction of RNA polymerase with forked DNA: Evidence for two kinetically significant intermediates on the pathway to the final complex

RNA polymerase forms competitor-resistant complexes with “forked DNA” templates that are double-stranded from the −35 promoter region through the first base pair of the −10 region, with an additional unpaired A at the 3′ end of the nontemplate strand. These types of substrates were introduced recently as model templates for the study of DNA–protein interactions occurring in the early stages of the formation of RNA polymerase–promoter open complexes. We have performed kinetic and equilibrium measurements of interactions of wild-type and mutant RNA polymerases bearing substitutions in the σ(70) initiation factor, with forked DNA of wild-type and mutant sequence. Our observations reveal that formation of a competitor-resistant complex between RNA polymerase and forked DNA, similar to the formation of open complexes at promoters, is a multistep process, and some of the sequentially formed intermediates along the two pathways share common properties. This work establishes, for the forked template, progression through these intermediates in the absence of downstream DNA and validates the use of forked DNA to determine the effects of changes in promoter or RNA polymerase sequence on the process of open complex formation.