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NTPase activities of HelD and release of HelD from RNAP a, b Comparison of NTPase activities of free HelD and its complexes with RNAP. ATP/GTP hydrolyzing activity of free HelD was set as 100%. ATP hydrolysis (a) is stimulated upon complex formation, whereas GTP hydrolysis (b) remains almost unchanged. Control measurements for individual complex components are shown. The bars show averages from three biological replicates, the error bars are ±SD, the dots represent individual experiments (also in panels d–g). c A scheme depicting the HelD release assay: His–RNAP was reconstituted into three different complexes, each containing combinations of HelD (cayn), σA (purple) and RbpA (yellow). The RNAP complexes were then allowed to bind to magnetic beads. The amount of HelD released, with or without addition of other factors (in panels d–g) was determined by Coomassie blue-stained SDS-PAGE gels and densitometry. d Effect of 1 mM ATP, GTP, or CTP on HelD release. In panels d–g, representative primary data are shown above the graph. Zero (Ø) shows HelD release without the addition of other factors. For this and experiments (e, g), the His–RNAP complex containing HelD, σA, and RbpA was used (depicted within the dashed box in c). The amount of HelD released from RNAP–σA–RbpA–HelD by the addition of ATP was set as 1 (also in other panels). A second primary data example is shown in Supplementary Fig. 14 together with a calibration curve used as quantification control. e, Effect of ATP analogs on HelD release. RNAP complexes were reconstituted as described in panel c with four HelD variants: WT-HelD (wild type), HelDσA-INT, HelDA-HYDRO, and HelDA-BIND (for definition of the mutants see Supplementary Fig. 16). Subsequently, 1 mM each of ATP, N-ATP, or ATPγS was added to the preformed RNAP complex attached to the magnetic beads and release of HelD from the complex was observed. f Release of HelD from the three types of complexes (c) induced with 1 mM ATP. g Effect of two forms of DNA and/or ATP on HelD release. CC, closed complex us-fork promoter DNA. OC open complex DNA with artificially opened transcription bubble. Source data are provided as a Source Data file.
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Mycobacterial HelD is a transcription factor that recycles stalled RNAP by dissociating it from nucleic acids and, if present, from the antibiotic rifampicin. The rescued RNAP, however, must disengage from HelD to participate in subsequent rounds of transcription. The mechanism of release is unknown. We show that HelD from Mycobacterium smegmatis f...
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The genomes of human gut bacteria in the genus Bacteroides include numerous operons for biosynthesis of diverse capsular polysaccharides (CPSs). The first two genes of each CPS operon encode a locus-specific paralog of transcription elongation factor NusG (called UpxY), which enhances transcript elongation, and a UpxZ protein that inhibits noncogna...
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... It is tempting to hypothesize about the regulatory advantage provided by the N-terminal extension into Mtb's ability to balance transcriptional efficiency with adaptability across different environmental contexts. While the exact molecular mechanisms remain to be elucidated, possibilities include modulation of interactions with core RNAP subunits and sequence-specific lineage insertions (74,109), or other regulatory proteins (110,111). Future studies will be needed to determine more specific mechanisms underlying σ A 's enhanced output to offer insights into the N-terminal extension's role in the biology of Mtb. ...
The regulation of ribosomal RNA (rRNA) is closely tied to nutrient availability, growth phase, and global gene expression, serving as a key factor in bacterial adaptability and pathogenicity. Mycobacterium tuberculosis ( Mtb ) stands out from other species with a single ribosomal operon controlled by two promoters: rrnA P3 and rrnA P1 and a high ratio of sigma (σ) factors to genome size. While the primary σ factor σ A is known to drive ribosomal transcription, the alternative σ factor σ B has been proposed to contribute to the transcription of housekeeping genes, including rRNA under a range of conditions. However, σ B 's precise role remains unclear. Here, we quantify steady-state rates in reconstituted transcription reactions and establish that σ A -mediated transcription from rrnA P3 dominates rRNA production by almost two orders of magnitude with minimal contributions from σ B holoenzymes and/or rrnA P1 under all conditions tested. We measure and compare the kinetics of individual initiation steps for both holoenzymes which, taken together with the steady-state rate measurements, lead us to a model where σ B holoenzymes exhibit slower DNA unwinding and slower holoenzyme recycling. Our data further demonstrate that the transcription factors CarD and RbpA reverse or buffer the stimulatory effect of negative superhelicity on σ A and σ B holoenzymes respectively. Lastly, we show that a major determinant of σ A 's increased activity is due to its N-terminal 205 amino acids. Taken together, our data reveal the intricate interplay of promoter sequence, σ factor identity, DNA superhelicity, and transcription factors in shaping transcription initiation kinetics and, by extension, the steady-state rates of rRNA production in Mtb.
... CarD and RbpA associate with the RNAP during transcription initiation and stabilize the open promoter complex (Hu et al, 2012;Hubin et al, 2017a;Rammohan et al, 2016;Rammohan et al, 2015;Srivastava et al, 2013;Stallings et al., 2009). Recently, HelD was identified as a protein that binds to non-transcribing RNAP and can interact with RNAP in complex with RbpA and the primary mycobacterial factor, A (Kouba et al, 2020;Kovaľ et al, 2024). ...
... HelD has been reported to restore transcription inhibited by rifampicin in vitro (Hurst-Hess et al., 2022;Kovaľ et al., 2024;Surette et al., 2022). In the presence of rifampicin, mRNA levels of three selected highly expressed genes (Ms1, rpoB, and sigA) were more reduced in ...
... /2024 The ChIP-seq data showed that RNAP peaks and σ A /σ B peaks tend to be higher when they overlap with HelD peaks (Fig. 4A and B). This correlation indicates that HelD is recruited to DNA by RNAP and/or σ A /σ B , which is consistent with the recently revealed RNAP-σ A -RbpA-HelD-DNA cryo-EM structure (Kovaľ et al., 2024). The presence of HelD correlates with increased gene expression also on a subset of tRNA genes. ...
HelD protein, also named HelR (encoded by MSMEG_2174 in Mycobacterium smegmatis), interacts with mycobacterial RNA polymerase (RNAP) and affects rifampicin resistance in Mycobacterium abscessus. Here, we provide data on rifampicin resistance and helD presence in the genomes of other clinically relevant nontuberculous mycobacteria.
We show that helD is primarily found in rapidly growing mycobacteria, such as M. smegmatis, where we detected HelD at a subset of promoters that can also associate with CarD and RbpA. Transcriptome analysis of a helD deletion strain using RNA-seq revealed that HelD enhances gene expression during exponential growth and decreases it in stationary phase, during which we observed reduced levels of CarD, RbpA, and GTP, the initiation nucleotide for the majority of M. smegmatis transcripts. We propose a model in which HelD releases abortive RNAP complexes and confirm that HelD dissociates RNAP from the promoter in vitro. HelD not only helps mycobacteria overcome rifampicin treatment but also supports efficient transcription during rapid growth, which indicates a dual role of this transcription regulator.
