Structures of RNA Polymerase Closed and Intermediate Complexes Reveal Mechanisms of DNA Opening and Transcription Initiation

Abstract

SummaryGene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo.

Response regulator VemR regulates the transcription of flagellar rod gene flgG by interacting with σ54 factor RpoN2 in Xanthomonas citri ssp. citri

“…RpoN–RNAP is unable to spontaneously isomerize from a closed complex to a transcriptionally competent open complex (Xu and Hoover, ). As an additional step before transcription initiation, the closed complex interacts with a transcriptional activator through the N‐terminus (Glyde et al ., ). These transcriptional activators usually bind at least 100 bp upstream of the promoter site, and DNA looping is required for the activator to contact the closed complex and catalyse the formation of the open promoter complex (Morett and Segovia, ).…”

An Introduction to the Structure and Function of the Catalytic Core Enzyme of Escherichia coli RNA Polymerase

“…The near-atomic resolution X-ray crystal structure of E. coli RNAP was determined first as holoenzyme containing σ 70 (Murakami, 2013). X-ray crystal and cryo-electron microscopy (cryo-EM) structures of E. coli RNAP are now available in several forms such as holoenzymes containing alternative σ factors (Liu et al, 2016; Yang et al, 2015), promoter DNA complexes (Glyde et al, 2017; Zuo and Steitz, 2015), elongation complex (Kang et al, 2017), in complex with transcription factors (Bae et al, 2013; Liu et al, 2017; Molodtsov et al, 2018), and with inhibitors/antibiotics (Bae et al, 2015; Chen et al, 2017; Degen et al, 2014; Molodtsov et al, 2015; Molodtsov et al, 2013), providing details of the structure and function of E. coli RNAP transcription and regulation.…”

Acclimation of bacterial cell state for high-throughput enzyme engineering using a DmpR-dependent transcriptional activation system

“…After the ligand binds the A domain, ATP hydrolysis by the released C domain triggers DmpR oligomerization and binding to promoter upstream regions. The conserved loop of ATPase interacts with the −12 DNA region of the promoter with DNA bending by the IHF and helps melt the DNA double strand to initiate transcription 8,9 . Along with DmpR as the bEBP, σ 54 is another important factor affecting the transcriptional initiation of the reporter gene in the DmpR-GESS.…”

“…TFs in cellular networks naturally respond to specific intracellular or extracellular molecules, followed by feedforward or feedback control for transcriptional regulation. In bacteria, sigma factor (σ) is an initial TF protein that binds the promoter DNA region, associates with core RNA polymerase (RNAP; subunits: α 2 , β, β′ and ω) and melts the DNA double strand for transcription initiation [6][7][8][9] . Among all TFs, sigma 54 (σ 54 , rpoN)-dependent TF would be a promising sensory protein in the development of biosensors given its well-documented response to specific molecules under starvation conditions 10,11 .…”