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

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hao-Hong Pei ◽  
Tarek Hilal ◽  
Zhuo A. Chen ◽  
Yong-Heng Huang ◽  
Yuan Gao ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Genome Stability ◽  
Slow Growth ◽  
Coiled Coil ◽  
Main Channel ◽  
Dependent Manner ◽  
Secondary Channel

AbstractCellular 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.

An energy charge sensor for balancing RNA polymerase recycling and hibernation

2020 ◽  
Author(s):  
Markus Wahl ◽  
Hao-Hong Pei ◽  
Tarek Hilal ◽  
Zhuo Chen ◽  
Yong-Heng Huang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Active Site ◽  
Genome Stability ◽  
Energy Levels ◽  
Energy Charge ◽  
Coiled Coil ◽  
Main Channel ◽  
Rna Polymerase I ◽  
Secondary Channel ◽  
Polymerase I

Abstract Cellular RNA polymerases can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, or enter dormancy. How RNA polymerase recycling into active states is achieved and balanced with quiescence remains elusive. We structurally analyzed Bacillus subtilis RNA polymerase bound to the NTPase HelD. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNA polymerase secondary channel, dismantling the active site and displacing RNA; a unique helical protrusion inserts into the main channel, prying β and β’ subunits apart and dislodging DNA, aided by the δ subunit. HelD release depends on ATP, and a dimeric structure resembling hibernating RNA polymerase I suggests that HelD can induce dormancy at low energy levels. Our results reveal an ingenious mechanism by which active RNA polymerase pools are adjusted in response to the nutritional state.

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

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomáš Kouba ◽  
Tomáš Koval’ ◽  
Petra Sudzinová ◽  
Jiří Pospíšil ◽  
Barbora Brezovská ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Rna Synthesis ◽  
Environmental Changes ◽  
Transcription Elongation ◽  
Specific Interactions ◽  
Inactive State ◽  
Transcription Machinery

AbstractRNA 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.

scholarly journals TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

2017 ◽  
Vol 114 (28) ◽  
pp. E5539-E5548 ◽  
Author(s):  
Saumya Gopalkrishnan ◽  
Wilma Ross ◽  
Albert Y. Chen ◽  
Richard L. Gourse
Keyword(s):  
Active Site ◽  
Transcription Initiation ◽  
Coiled Coil ◽  
Global Regulator ◽  
Secondary Channel ◽  
Global Regulators ◽  
Extrachromosomal Elements ◽  
Distant Homolog ◽  
Active Site Region

TheEscherichia coliF element-encoded protein TraR is a distant homolog of the chromosome-encoded transcription factor DksA. Here we address the mechanism by which TraR acts as a global regulator, inhibiting some promoters and activating others. We show that TraR regulates transcription directly in vitro by binding to the secondary channel of RNA polymerase (RNAP) using interactions similar, but not identical, to those of DksA. Even though it binds to RNAP with only slightly higher affinity than DksA and is only half the size of DksA, TraR by itself inhibits transcription as strongly as DksA and ppGpp combined and much more than DksA alone. Furthermore, unlike DksA, TraR activates transcription even in the absence of ppGpp. TraR lacks the residues that interact with ppGpp in DksA, and TraR binding to RNAP uses the residues in the β′ rim helices that contribute to the ppGpp binding site in the DksA–ppGpp–RNAP complex. Thus, unlike DksA, TraR does not bind ppGpp. We propose a model in which TraR mimics the effects of DksA and ppGpp together by binding directly to the region of the RNAP secondary channel that otherwise binds ppGpp, and its N-terminal region, like the coiled-coil tip of DksA, engages the active-site region of the enzyme and affects transcription allosterically. These data provide insights into the function not only of TraR but also of an evolutionarily widespread and diverse family of TraR-like proteins encoded by bacteria, as well as bacteriophages and other extrachromosomal elements.

scholarly journals Sulfate-Dependent Repression of Genes That Function in Organosulfur Metabolism in Bacillus subtilis Requires Spx

2005 ◽  
Vol 187 (12) ◽  
pp. 4042-4049 ◽  
Author(s):  
Kyle N. Erwin ◽  
Shunji Nakano ◽  
Peter Zuber
Keyword(s):  
Oxidative Stress ◽  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Transcriptional Control ◽  
Sulfur Metabolism ◽  
Growth Conditions ◽  
Sulfur Source ◽  
Dependent Manner ◽  
Α Subunit ◽  
A Genome

ABSTRACT Oxidative stress in Bacillus subtilis results in the accumulation of Spx protein, which exerts both positive and negative transcriptional control over a genome-wide scale through its interaction with the RNA polymerase α subunit. Previous microarray transcriptome studies uncovered a unique class of genes that are controlled by Spx-RNA polymerase interaction under normal growth conditions that do not promote Spx overproduction. These genes were repressed by Spx when sulfate was present as a sole sulfur source. The genes include those of the ytmI, yxeI, and ssu operons, which encode products resembling proteins that function in the uptake and desulfurization of organic sulfur compounds. Primer extension and analysis of operon-lacZ fusion expression revealed that the operons are repressed by sulfate and cysteine; however, Spx functioned only in sulfate-dependent repression. Both the ytmI operon and the divergently transcribed ytlI, encoding a LysR-type regulator that positively controls ytmI operon transcription, are repressed by Spx in sulfate-containing media. The CXXC motif of Spx, which is necessary for redox sensitive control of Spx activity in response to oxidative stress, is not required for sulfate-dependent repression. The yxeL-lacZ and ssu-lacZ fusions were also repressed in an Spx-dependent manner in media containing sulfate as the sole sulfur source. This work uncovers a new role for Spx in the control of sulfur metabolism in a gram-positive bacterium under nonstressful growth conditions.

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

2020 ◽  
Author(s):  
Tomáš Kouba ◽  
Tomáš Koval’ ◽  
Petra Sudzinová ◽  
Jiří Pospíšil ◽  
Barbora Brezovská ◽  
...  
Keyword(s):  
Dna Binding ◽  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Rna Synthesis ◽  
Environmental Changes ◽  
Specific Interactions ◽  
Σ Factor ◽  
Polymerase Binding

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.

scholarly journals Mutational analysis of Escherichia coli GreA protein reveals new functional activity independent of antipause and lethal when overexpressed

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Llorenç Fernández-Coll ◽  
Katarzyna Potrykus ◽  
Michael Cashel ◽  
Carlos Balsalobre
Keyword(s):  
Rna Polymerase ◽  
Binding Sites ◽  
Mutational Analysis ◽  
Coiled Coil ◽  
Secondary Channel ◽  
Reporter System ◽  
E Coli ◽  
Functional Studies ◽  
The Family ◽  
Pause Site

Abstract There is a growing appreciation for the diverse regulatory consequences of the family of proteins that bind to the secondary channel of E. coli RNA polymerase (RNAP), such as GreA, GreB or DksA. Similar binding sites could suggest a competition between them. GreA is characterised to rescue stalled RNAP complexes due to its antipause activity, but also it is involved in transcription fidelity and proofreading. Here, overexpression of GreA is noted to be lethal independent of its antipause activity. A library of random GreA variants has been used to isolate lethality suppressors to assess important residues for GreA functionality and its interaction with the RNA polymerase. Some mutant defects are inferred to be associated with altered binding competition with DksA, while other variants seem to have antipause activity defects that cannot reverse a GreA-sensitive pause site in a fliC::lacZ reporter system. Surprisingly, apparent binding and cleavage defects are found scattered throughout both the coiled-coil and globular domains. Thus, the coiled-coil of GreA is not just a measuring stick ensuring placement of acidic residues precisely at the catalytic centre but also seems to have binding functions. These lethality suppressor mutants may provide valuable tools for future structural and functional studies.

scholarly journals DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

2015 ◽  
Vol 112 (50) ◽  
pp. E6862-E6871 ◽  
Author(s):  
Andrey Parshin ◽  
Anthony L. Shiver ◽  
Jookyung Lee ◽  
Maria Ozerova ◽  
Dina Schneidman-Duhovny ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Active Site ◽  
Amino Acid Starvation ◽  
Cross Linking ◽  
Molecular Function ◽  
Secondary Channel ◽  
Global Response ◽  
Microbial Life ◽  
Active Site Region

Sensing and responding to nutritional status is a major challenge for microbial life. In Escherichia coli, the global response to amino acid starvation is orchestrated by guanosine-3′,5′-bisdiphosphate and the transcription factor DksA. DksA alters transcription by binding to RNA polymerase and allosterically modulating its activity. Using genetic analysis, photo–cross-linking, and structural modeling, we show that DksA binds and acts upon RNA polymerase through prominent features of both the nucleotide-access secondary channel and the active-site region. This work is, to our knowledge, the first demonstration of a molecular function for Sequence Insertion 1 in the β subunit of RNA polymerase and significantly advances our understanding of how DksA binds to RNA polymerase and alters transcription.

scholarly journals Structural basis of transcription arrest by coliphage HK022 Nun in an Escherichia coli RNA polymerase elongation complex

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jin Young Kang ◽  
Paul Dominic B Olinares ◽  
James Chen ◽  
Elizabeth A Campbell ◽  
Arkady Mustaev ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Terminal Segment ◽  
Structural Basis ◽  
Elongation Complex ◽  
Structural Framework ◽  
Cryo Electron Microscopy ◽  
Active Site Cleft

Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λ DNA. To provide a structural framework to understand how Nun blocks RNAP translocation, we determined structures of Escherichia coli RNAP ternary elongation complexes (TECs) with and without Nun by single-particle cryo-electron microscopy. Nun fits tightly into the TEC by taking advantage of gaps between the RNAP and the nucleic acids. The C-terminal segment of Nun interacts with the RNAP β and β’ subunits inside the RNAP active site cleft as well as with nearly every element of the nucleic acid scaffold, essentially crosslinking the RNAP and the nucleic acids to prevent translocation, a mechanism supported by the effects of Nun amino acid substitutions. The nature of Nun interactions inside the RNAP active site cleft suggests that RNAP clamp opening is required for Nun to establish its interactions, explaining why Nun acts on paused TECs.

scholarly journals Regulation of transcriptional pausing through the secondary channel of RNA polymerase

2016 ◽  
Vol 113 (31) ◽  
pp. 8699-8704 ◽  
Author(s):  
Daria Esyunina ◽  
Aleksei Agapov ◽  
Andrey Kulbachinskiy
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Active Site ◽  
Rna Structure ◽  
Deinococcus Radiodurans ◽  
Genetic Regulation ◽  
Secondary Channel ◽  
Trigger Loop ◽  
Transcriptional Pausing ◽  
External Stimuli

Transcriptional pausing has emerged as an essential mechanism of genetic regulation in both bacteria and eukaryotes, where it serves to coordinate transcription with other cellular processes and to activate or halt gene expression rapidly in response to external stimuli. Deinococcus radiodurans, a highly radioresistant and stress-resistant bacterium, encodes three members of the Gre family of transcription factors: GreA and two Gre factor homologs, Gfh1 and Gfh2. Whereas GreA is a universal bacterial factor that stimulates RNA cleavage by RNA polymerase (RNAP), the functions of lineage-specific Gfh proteins remain unknown. Here, we demonstrate that these proteins, which bind within the RNAP secondary channel, strongly enhance site-specific transcriptional pausing and intrinsic termination. Uniquely, the pause-stimulatory activity of Gfh proteins depends on the nature of divalent ions (Mg2+ or Mn2+) present in the reaction and is also modulated by the nascent RNA structure and the trigger loop in the RNAP active site. Our data reveal remarkable plasticity of the RNAP active site in response to various regulatory stimuli and highlight functional diversity of transcription factors that bind inside the secondary channel of RNAP.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

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