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

ABSTRACT: RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. We show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP 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 releases RNAP from nonfunctional complexes with nucleic acids.

SUBMITTER: Kouba T

PROVIDER: S-EPMC7749160 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Molecular insights into RNA polymerase recycling by mycobacterial HelD

Project description:Mycobacterial HelD is a transcription factor that binds RNA polymerase (RNAP) and thereby rescues it from stalled transcription complexes. HelD also protects RNAP against the antibiotic rifampicin. HelD, however, must be released from the rescued RNAP to participate in the next round of the transcription cycle. The exact mechanism of this release is unknown. Here we show that HelD from Mycobacterium smegmatis can form a complex with RNAP, together with σA primary sigma factor, and RbpA, but not CarD, transcription factors. Using cryo-EM, we solved a series of RNAP-σA-RbpA-HelD structures with or without promoter DNA. These snapshots capture the involvement of HelD in transcription initiation, describes mechanistic aspects of HelD release, and its protective effect against rifampicin. Biochemical evidence supports these findings, defines the role of ATP binding/hydrolysis to/by HelD in the process, and functionally confirms the rifampicin-protective effect of HelD. Taken together, HelD assists an alternative pathway of transcription initiation.

2024-10-08 | PXD046632 | Pride

EWSR1's visual modalities are defined by its association with nucleic acids and RNA polymerase II.

Project description:We report systematic analysis of endogenous EWSR1's cellular organization. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Interestingly, EWSR1 and FUS, another FET protein, exhibit distinct spatial organizations. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.

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Bacillus subtilis HelD, an RNA Polymerase Interacting Helicase, Forms Amyloid-Like Fibrils.

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Characterization of HelD, an interacting partner of RNA polymerase from Bacillus subtilis.

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Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD.

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Project description:A polymerase ribozyme can be used to label the 3' end of RNA or DNA molecules by incorporating a variety of functionalized nucleotide analogs. Guided by a complementary template, the ribozyme adds a single nucleotide that may contain a fluorophore, biotin, azide or alkyne moiety, thus enabling the detection and/or capture of selectively labeled materials. Employing a variety of commercially available nucleotide analogs, efficient labeling was demonstrated for model RNAs and DNAs, human microRNAs and natural tRNA.

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Cryo-EM structure of Bacillus subtilis RNA Polymerase in complex with HelD

Project description: Not available

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The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling.

Project description:Cellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD complexes. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the β and β' subunits apart and, aided by δ, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP-dependent manner. HelD abundance during slow growth and a dimeric (RNAP-δ-HelD)2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cues.

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