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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

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

Project description:Bacterial RNA polymerase (RNAP) is an essential multisubunit protein complex required for gene expression. Here, we characterize YvgS (HelD) from Bacillus subtilis, a novel binding partner of RNAP. We show that HelD interacts with RNAP-core between the secondary channel of RNAP and the alpha subunits. Importantly, we demonstrate that HelD stimulates transcription in an ATP-dependent manner by enhancing transcriptional cycling and elongation. We demonstrate that the stimulatory effect of HelD can be amplified by a small subunit of RNAP, delta. In vivo, HelD is not essential but it is required for timely adaptations of the cell to changing environment. In summary, this study establishes HelD as a valid component of the bacterial transcription machinery.

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

Project description:HelD, an RNA polymerase binding protein from Bacillus subtilis, stimulates transcription and helps in timely adaptation of cells under diverse environmental conditions. At present, no structural information is available for HelD. In the current study, we performed size exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS) which suggests that HelD is predominantly monomeric and globular in solution. Using combination of size exclusion chromatography and analytical ultracentrifugation, we also show that HelD has a tendency to form higher order oligomers in solution. CD experiments suggest that HelD has both ?-helical (?35%) and ? sheet (?26%) secondary structural elements. Thermal melting experiments suggest that even at 90°C, there is only about 30% loss in secondary structural contents with Tm of 44°C. However, with the increase in temperature, there was a gain in the ?-sheet content and significant irreversible loss of ?-helical content. Using a combination of X-ray fiber diffraction analysis, and dye based assays including Thioflavin-T based fluorescence and Congo red binding assays, we discovered that HelD forms amyloid-like fibrils at physiologically relevant conditions in vitro. Using confocal imaging, we further show that HelD forms amyloid inclusions in Escherichia coli. Bioinformatics-based sequence analysis performed using three independent web-based servers suggests that HelD has more than 20 hot-spots spread across the sequence that may aid the formation of amyloid-like fibrils. This discovery adds one more member to the growing list of amyloid or amyloid-like fibril forming cytosolic proteins in bacteria. Future studies aimed at resolving the function of amyloid-like fibrils or amyloid inclusions may help better understand their role, if any, in the bacterial physiology.

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

Project description:Efficient control of transcription is essential in all organisms. In bacteria, where DNA replication and transcription occur simultaneously, the replication machinery is at risk of colliding with highly abundant transcription complexes. This can be exacerbated by the fact that transcription complexes pause frequently. When pauses are long-lasting, the stalled complexes must be removed to prevent collisions with either another transcription complex or the replication machinery. HelD is a protein that represents a new class of ATP-dependent motor proteins distantly related to helicases. It was first identified in the model Gram-positive bacterium Bacillus subtilis and is involved in removing and recycling stalled transcription complexes. To date, two classes of HelD have been identified: one in the low G+C and the other in the high G+C Gram-positive bacteria. In this work, we have undertaken the first comprehensive investigation of the phylogenetic diversity of HelD proteins. We show that genes in certain bacterial classes have been inherited by horizontal gene transfer, many organisms contain multiple expressed isoforms of HelD, some of which are associated with antibiotic resistance, and that there is a third class of HelD protein found in Gram-negative bacteria. In summary, HelD proteins represent an important new class of transcription factors associated with genome maintenance and antibiotic resistance that are conserved across the Eubacterial kingdom.

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3'-End labeling of nucleic acids by a polymerase ribozyme.

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.

| S-EPMC6158495 | biostudies-literature

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

Project description: Not available

| EMPIAR-11052 | biostudies-other

Design Considerations for RNA Spherical Nucleic Acids (SNAs).

Project description:Ribonucleic acids (RNAs) are key components in many cellular processes such as cell division, differentiation, growth, aging, and death. RNA spherical nucleic acids (RNA-SNAs), which consist of dense shells of double-stranded RNA on nanoparticle surfaces, are powerful and promising therapeutic modalities because they confer advantages over linear RNA such as high cellular uptake and enhanced stability. Due to their three-dimensional shell of oligonucleotides, SNAs, in comparison to linear nucleic acids, interact with the biological environment in unique ways. Herein, the modularity of the RNA-SNA is used to systematically study structure-function relationships in order to understand how the oligonucleotide shell affects interactions with a specific type of biological environment, namely, one that contains serum nucleases. We use a combination of experiment and theory to determine the key architectural properties (i.e., sequence, density, spacer moiety, and backfill molecule) that affect how RNA-SNAs interact with serum nucleases. These data establish a set of design parameters for SNA architectures that are optimized in terms of stability.

| S-EPMC5034328 | biostudies-literature

Double-headed nucleotides as xeno nucleic acids: information storage and polymerase recognition.

Project description:Xeno nucleic acids (XNAs) are artificial genetic systems based on sugar-modified nucleotides. Herein, we investigate double-headed nucleotides as a new XNA. A new monomer, AT, is presented, and together with previous double-headed nucleotide monomers, new nucleic acid motifs consisting of up to five consecutive A·T base pairs have been obtained. Sections composed entirely of double-headed nucleotides are well-tolerated within a DNA duplex and can condense the genetic information. For instance, a 13-mer duplex is condensed to an 11-mer modified duplex containing four double-headed nucleotides while simultaneously improving duplex thermal stability with +14.0 °C. Also, the transfer of information from double-headed to natural nucleotides by DNA polymerases has been examined. The first double-headed nucleoside triphosphate was prepared but could not be recognized and incorporated by the tested DNA polymerases. On the other hand, it proved possible for Therminator DNA polymerase to transfer the information of a double-headed nucleotide in a template sequence to natural DNA under controlled conditions.

| S-EPMC7517788 | biostudies-literature

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.

| S-EPMC7749165 | biostudies-literature

Messenger RNA enrichment using synthetic oligo(T) click nucleic acids.

Project description:Enrichment of mRNA is a key step in a number of molecular biology techniques, particularly in the rapidly growing field of transcriptomics. Currently, mRNA is isolated using oligo(thymine) DNA (oligo(dT)) immobilized on solid supports, which binds to the poly(A) tail of mRNA to pull the mRNA out of solution through the use of magnets or centrifugal filters. Here, a simple method to isolate mRNA by complexing it with synthetic click nucleic acids (CNAs) is described. Oligo(T) CNA bound efficiently to mRNA, and because of the insolubility of CNA in water, >90% of mRNA was readily removed from solution using this method. Simple washing, buffer exchange, and heating steps enabled mRNA's enrichment from total RNA, with a yield of 3.1 ± 1.5% of the input total RNA by mass, comparable to the yield from commercially available mRNA enrichment beads. Further, the integrity and activity of mRNA after CNA-facilitated pulldown and release was evaluated through two assays. In vitro translation of EGFP mRNA confirmed the translatability of mRNA into functional protein and RT-qPCR was used to amplify enriched mRNA from total RNA extracts and compare gene expression to results obtained using commercially available products.

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