Structure, function, and regulation of the bacterial transcription cycle

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Project Summary Transcription is the major control point of gene expression and RNA polymerase (RNAP), conserved from bacteria to man, is the central enzyme of transcription. Our long term goal is to understand the mechanism of transcription and its regulation. Determining three-dimensional structures of RNAP and its complexes with DNA, RNA, and regulatory factors, is an essential step. We focus on highly characterized prokaryotic RNAPs. The basic elements of the transcription cycle, initiation, elongation, and termination, were elucidated through study of prokaryotes. A detailed structural and functional understanding of the entire transcription cycle is essential to explain the fundamental control of gene expression and to target RNAP with small-molecule antibiotics. Moreover, a complete understanding of how a complex, molecular machine uses binding and chemical energy to effect conformational changes that drive the cycle, and how regulators modulate the cycle, is of fundamental interest. The transcription cycle is marked by a series of stable complexes (core è holo è RPo è EC) that interconvert through transient intermediates. The transitions between stable states are points of heavy regulation that are poorly understood due to the lack of structural information. Major transitions include: Holoenzyme + promoter DNA è open promoter complex (initiation) Open promoter complex è elongation complex (promoter escape, s dissociation) Elongation complex è core RNAP + DNA + completed RNA transcript (termination) Each of these transitions is characterized by unstable, transient intermediates that are extremely challenging for structural biology. Cryo-electron microscopy (cryo-EM) has emerged as a powerful method to visualize these transient states. We are combining cryo-EM with other approaches to mechanistically and structurally characterize transient intermediates that govern transitions in the bacterial transcription cycle, including promoter melting, the initiation to elongation transition, and transcription termination. These findings will provide insight into the behavior of macromolecular machines throughout biology.