MOLECULAR MECHANISMS OF PROMOTER BINDING BY ESCHERICHIA COLI RNA POLYMERASE

Abstract

DNA strand and enzyme subunit specificities involved in the interaction between E. coli RNA polymerase and T7 DNA were studied by the photocrosslinking technique. In nonspecific enzyme-DNA complexes, subunits (sigma), (beta), and (beta)' were crosslinked to both strands of the DNA while in specific enzyme-promoter complexes, only (sigma) and (beta) subunits were crosslinked. The (sigma) subunit was crosslinked preferentially to the nonsense strand at promoter sites, with no such strand specificity observed for the (beta) subunit. These results indicate that the interaction between RNA polymerase and DNA template differs at promoter and nonspecific sites. These differences could provide the structural basis for the recognition of the few promoter sites on a template by RNA polymerase.;In order to gain a more dynamic picture of the interaction process, the stopped-flow rapid photocrossing technique was developed. Rapid mixing was used to synchronize the interaction process while rapid photocrossing was used to stabilize or freeze the structure of the protein-DNA complexes at various stages of formation. This technique was utilized to measure the kinetics of the binding process, provide a dynamic structural picture of the binding reaction, and to elucidate the mechanism used by RNA polymerase to search for the promoter sites.;Binding of RNA polymerase to DNA occurs in at least two phases, the rapid initial nonspecific binding followed by a slower promoter search step. The initial binding is a diffusion limited reaction which occurs randomly with respect to subunit contacts and position of binding along the DNA molecule. Once bound, the enzyme is effectively trapped within that DNA molecule by the "domain" effect. Thus, RNA polymerase searches for the promoter sites predominantly by a series of intradomain transfer events. It was concluded that the overall mechanism of promoter search is linear diffusion along the DNA strand. These results are in accord with the notion that the entire DNA molecule serves to trap and guide the diffusion of RNA polymerase during promoter search.