Effects of Discriminator Changes on Open Complex Formation, Stabilization, and Transcription Initiation

Abstract

The discriminator is the region of promoter DNA between the key −10 recognition region for RNA polymerase (RNAP) and the transcription start site (TSS). The length, sequences of the discriminator, and the interactions between RNAP mobile elements and promoter DNA the discriminator direct, are major determinants of the lifetime and the structure of the RNAP-promoter open complex (OC). The OC lifetime and rate of formation can both vary by ∼4 orders of magnitude for different promoter sequences, and together regulate the rate of initiation and escape of RNAP from these promoters. We are investigating a series of discriminators including those of the long-lived λPR OC (GGTTGC) and the short-lived rrnBP1 OC (GCGCCACC), in the context of the upstream λPR promoter and a modified λPR transcribed region. Fast kinetic methods with filter binding detection are used to determine overall rate constants for open complex formation and dissociation, and to dissect contributions to these rate constants from initial equilibria involving closed or open complexes (CC or OC), and the rate-determining isomerizations step between them. Our results to date indicate that the discriminator affects primarily the steps that stabilize the initial OC, and has only limited effects on the DNA closing rate constant or on the overall second order rate constant for OC formation. From previous studies, interactions of in-cleft (ss) DNA and downstream (ds) DNA with RNAP change in the process of stabilization of the initial OC. Regions 1.1 and 1.2 of σ70 and downstream mobile elements of core RNAP are involved, and the orientation of the downstream duplex is changed. We are investigating these interactions and conformational changes for different discriminators in more detail, and the relationship to initiation and promoter escape of RNAP.