RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding

Abstract The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNA...

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Veröffentlicht in:Nucleic acids research 2021-03, Vol.49 (5), p.2790-2802
Hauptverfasser: Mazumder, Abhishek, Wang, Anna, Uhm, Heesoo, Ebright, Richard H, Kapanidis, Achillefs N
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container_end_page 2802
container_issue 5
container_start_page 2790
container_title Nucleic acids research
container_volume 49
creator Mazumder, Abhishek
Wang, Anna
Uhm, Heesoo
Ebright, Richard H
Kapanidis, Achillefs N
description Abstract The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3–0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes > 1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.
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subjects Cations, Divalent
Cations, Monovalent
DNA - metabolism
DNA-Directed RNA Polymerases - chemistry
DNA-Directed RNA Polymerases - metabolism
Escherichia coli - enzymology
Fluorescence Resonance Energy Transfer
Nucleic Acid Enzymes
Polyethylene Glycols - chemistry
Protein Conformation
title RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding
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