Toward a General Mechanism for Transcription Initiation

Abstract

At the λPR promoter, formation of initiation-competent open complexes involves a series of conformational changes in both RNA polymerase (RNAP) and promoter DNA after initial specific binding. The first set of these changes (described by equilibrium constant K1) bend the downstream duplex DNA into the cleft of RNAP. These reversible steps are followed by the rate-determining step (rate constant k2) in which the DNA is opened in the cleft using binding free energy. This initial open complex is unstable (lifetime ∼1 s) but is greatly stabilized by irreversible conformational changes (quantified by equilibrium constant K3) that reposition the nontemplate strand in the cleft and assemble downstream mobile elements (DME) of RNAP on the downstream duplex DNA, forming RPo (lifetime ∼1 day).In all three phases of this mechanism, large conformational changes in RNAP and/or promoter DNA take place, and allosteric communication occurs over large distances. However, the details are not yet understood for λPR or any other promoter. Are these mechanistic steps universal or promoter-specific? Which steps are the most important targets of regulation by promoter sequence, transcription factors, ligands, and solutes? What determines the efficiency of productive or abortive transcription?To address these questions, we have determined the effects of sequence and length variants of the λPR and T7A1 promoters and several RNAP variants on the kinetics of steps of the initiation mechanism. Taken together, our kinetic data suggest that the early and late steps of the mechanism are the most variable. We therefore propose that these steps are the targets of most cis- and trans-acting regulatory factors, while the opening/closing step is relatively unregulated. This is analogous to many enzyme mechanisms in which most regulation of catalytic velocity occurs in the early and late steps rather than the catalytic step itself.