RNA polymerase: A nexus of gene regulation

John D. Helmann
2009 Methods  
In Bacteria, transcription is catalyzed by a single RNA polymerase (RNAP) whose promoter selectivity and activity is governed by a wide variety of transcription factors. The net effect of these transcriptional regulators is to determine which genes are transcribed, and at what levels, under any specific growth condition. RNAP thus serves as a nexus of gene regulation that integrates the information coming from a variety of sensory systems to appropriately modulate gene expression. The
more » ... presented in this volume provide a set of tools and approaches for investigating the factors controlling RNAP activity at both individual promoters and on a genomic scale. This introductory chapter provides a brief overview of RNAP and the transcription cycle and introduces general principles of how the fundamental steps of transcription are influenced by both DNA (promoter) sequences and trans-acting factors. polymer complementary to specific regions of the DNA template. Like all macromolecular synthesis, RNA synthesis can be divided into three general phases: initiation, elongation, and termination [4] . Importantly, each of these phases can be a target of regulation. Bacterial RNAP is a multisubunit enzyme and consists of a core polymerase (abbreviated as E) containing the beta, beta', and two alpha subunits (together with one or more omega subunits; [5, 6] ) and a dissociable specificity factor known as sigma (σ) [7] . While the core RNAP (minimally,ββ'α 2 ) is competent for transcription elongation and termination, transcript initiation requires an associated σ subunit. The core+σ complex is designated holoenzyme. Many bacteria contain multiple types of σ factor and, therefore, multiple holoenzyme species [8, 9] . In E. coli, the primary σ is called σ 70 and is required for the transcription of most genes including those for many essential functions. So-called alternative σ factors often control specialized sets of genes that are required for specific cellular functions (e.g. motility and chemotaxis) or under stress conditions (e.g. heat shock). While most σ factors are structurally related to σ 70 , a second family of σ proteins known as σ 54 (or σ N ) also mediates promoter recognition of specialized sets of genes [10]. Promoter recognition and transcript initiation The first step in RNA synthesis, and the most common target for regulation, is initiation. The ability to identify start sites of transcription and initiate chains de novo distinguishes RNAP from the mechanistically related enzymes that synthesize DNA (which require a separate primase to generate a primer for elongation; [11] ). Since initiation is a frequent target for regulation, and is itself an exceedingly complex reaction, it is worth reviewing the key stages in this process. These can be defined as (i) promoter recognition, (ii) transcript initiation, and (iii) promoter clearance.
doi:10.1016/j.ymeth.2008.12.001 pmid:19070783 pmcid:PMC3022018 fatcat:yewbl34fkngvxbpskpdo3tvfxm