Domain structure of HelD, an interaction partner of <i>Bacillus subtilis </i><scp>RNA</scp> polymerase

Kovaĺ, T.; Sudzinová, Petra; Perháčová, Terézia; Trundová, Mária; Skálová, Tereza; Fejfarová, Karla; Šanderová, Hana; Krásný, Libor; Dušková, Jarmila; Dohnálek, Jan

doi:10.1002/1873-3468.13385

Cited by 8 publications

(16 citation statements)

References 36 publications

(43 reference statements)

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“…The presented structure confirms our previous prediction 9 where HelD, similarly to SF1 helicases, RapA and UvrD, contains a conserved Rossmann fold 1A-2A heterodimer. 1A-1 is connected with the N-terminal domain by the NG linker (HelD/145-173), which orders only in State I.…”

Section: Resultssupporting

confidence: 90%

“…One of these factors is HelD 5 , a protein similar to SF1 helicases 7 that associates with the RNAP core in the model gram-positive bacterium Bacillus subtilis ( Bsu ) where it was shown to be involved in transcriptional recycling 8 . HelD binds and hydrolyzes ATP and this is accompanied by conformational changes in the protein as demonstrated by SAXS experiments 9 . The absence of HelD from Bsu cells results in prolonged lag phase during outgrowth of stationary phase cells when diluted into fresh medium; overexpression of HelD then accelerates spore formation 10 .…”

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confidence: 99%

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Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

Kouba

Kovaĺ

Sudzinová

et al. 2020

Preprint

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SUMMARYRNA synthesis is central to life, and RNA polymerase depends on accessory factors for recovery from stalled states and adaption to environmental changes. Here we investigated the mechanism by which a helicase-like factor HelD recycles RNA polymerase. We report a cryo-EM structure of an unprecedented complex between the Mycobacterium smegmatis RNA polymerase and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNA polymerase channels that are responsible for DNA binding and substrate delivery to the active site, thereby locking RNA polymerase in an inactive state. We show that HelD prevents non-specific interactions between RNA polymerase and DNA and dissociates transcription elongation complexes, but does not inhibit RNA polymerase binding to the initiation σ factor. The liberated RNA polymerase can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that removes undesirable nucleic acids from RNA polymerase.

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Section: Resultssupporting

confidence: 90%

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confidence: 99%

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

Kouba

Kovaĺ

Sudzinová

et al. 2020

Preprint

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“…Superposition with the nucleic acids from the EC show that this positively-charged patch is in a position where it could interact with the downstream dsDNA (Extended Data Fig. 8a), consistent with nucleic acid-binding data 13 . It is also possible that this patch may be important for interaction with the unstructured negatively-charged C-terminal domain of δ which acts synergistically with HelD during transcription complex recycling 12 (see accompanying paper by Pei et al).…”

Section: Held Causes Major Conformational Changes In Rnapsupporting

confidence: 83%

“…Strikingly, the structure showed that HelD, which is located on the downstream side of RNAP, has two arm domains that penetrate deep into the primary and secondary channels of RNAP (clamp arm; CA, and secondary channel arm; SCA, respectively, Fig. 2b-d), which account for the strong HelD-RNAP interaction 12,13,35 . The native RNAP-HelD preparation also contained the RNAP ε subunit 19 and showed it bound on the downstream side of RNAP in a concave space between the two α, β, and β' subunits ( Fig.…”

Section: Structure Determination Of the Transcription Elongation Complexmentioning

confidence: 99%

Molecular Basis for RNA Polymerase-Dependent Transcription Complex Recycling By The Helicase-like Motor Protein HelD.

Newing

Oakley

Miller

et al. 2020

Preprint

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In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using cryo-electron microscopy and single-particle analysis, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the extraordinary conformational changes that occur in RNAP driven by HelD interaction. HelD has a unique 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a dramatic conformational change in the primary channel leading to removal and recycling of the stalled polymerase.

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“…HelD is widely distributed in Gram-positive bacteria and has superficial similarity to superfamily 1 (SF1) DNA helicases such as UvrD/PcrA, and its catalytic activity is ATP-dependent 12 . Lowresolution small-angle X-ray scattering data indicate that HelD undergoes an ATP-dependent conformational change and is capable of binding to DNA 13 , suggesting that these are important properties of HelD in transcription complex recycling. SF1 helicases UvrD/PcrA bind on the upstream side of RNAP, and are able to reverse-translocate it away from a site of DNA damage 6,14 .…”

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confidence: 99%

Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

et al. 2020

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In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.

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Domain structure of HelD, an interaction partner of Bacillus subtilis RNA polymerase

Cited by 8 publications

References 36 publications

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

Molecular Basis for RNA Polymerase-Dependent Transcription Complex Recycling By The Helicase-like Motor Protein HelD.

Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

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