Structures of Bacterial RNA Polymerase Complexes Reveal the Mechanism of DNA Loading and Transcription Initiation

Robert Glyde,Fuzhou Ye,Milija Jovanovic,Ioly Kotta-Loizou,Martin Buck,Xiaodong Zhang

doi:10.1016/j.molcel.2018.05.021

Abstract

SummaryGene transcription is carried out by multi-subunit RNA polymerases (RNAPs). Transcription initiation is a dynamic multi-step process that involves the opening of the double-stranded DNA to form a transcription bubble and delivery of the template strand deep into the RNAP for RNA synthesis. Applying cryoelectron microscopy to a unique transcription system using σ54 (σN), the major bacterial variant sigma factor, we capture a new intermediate state at 4.1 Å where promoter DNA is caught at the entrance of the RNAP cleft. Combining with new structures of the open promoter complex and an initial de novo transcribing complex at 3.4 and 3.7 Å, respectively, our studies reveal the dynamics of DNA loading and mechanism of transcription bubble stabilization that involves coordinated, large-scale conformational changes of the universally conserved features within RNAP and DNA. In addition, our studies reveal a novel mechanism of strand separation by σ54.

Highlights

Gene transcription is a fundamental cellular process carried out by the multi-subunit RNA polymerase (RNAP), which is conserved from bacteria to humans (Cramer, 2002; Werner, 2008)
The s54, which controls stress related genes including those involved in nitrogen fixation, nutrient starvation, infection, and other cellular stresses, forms a class of its own (Buck et al, 2000; Merrick, 1993). s54 recruits RNAP to its promoter sites via binding to the À12 and À24 consensus promoter sequences and forms a stable RPc that rarely spontaneously isomerizes to RPo
Using the unique properties of the s54 system, we have recently determined the structures of a bacterial RPc and one transcription intermediate complex (RPi), using the activator phage shock protein F (PspF) in complex with an ATP hydrolysis transition state analog, ADP.AlFx (Glyde et al, 2017)

Summary

Graphical Abstract

Glyde et al report three cryo-EM structures of bacterial transcription initiation complexes. These structures support a coupled DNA load and unwind model that involves an initial opening of the RNA polymerase clamp to facilitate DNA loading before it closes down to complete the loading, which leads to DNA unwinding. 2018, Molecular Cell 70, 1111–1120 June 21, 2018 a 2018 The Author(s).

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