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

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.

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

10.1101/2020.07.20.211821 ◽

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.

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

Nature Communications ◽

10.1038/s41467-020-20159-3 ◽

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.

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Mitotic repression of RNA polymerase II transcription is accompanied by release of transcription elongation complexes.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5791 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5791-5802 ◽

Cited By ~ 67

Author(s):

G G Parsons ◽

C A Spencer

Keyword(s):

Cell Cycle ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Synthesis ◽

Transcription Elongation ◽

Control Cell ◽

Specific Gene ◽

Mitotic Chromosomes ◽

Transcription Repression ◽

Rna Polymerase Ii Transcription

Nuclear RNA synthesis is repressed during the mitotic phase of each cell cycle. Although total RNA synthesis remains low throughout mitosis, the degree of RNA polymerase II transcription repression on specific genes has not been examined. In addition, it is not known whether mitotic repression of RNA polymerase II transcription is due to polymerase pausing or ejection of transcription elongation complexes from mitotic chromosomes. In this study, we show that RNA polymerase II transcription is repressed in mammalian cells on a number of specific gene regions during mitosis. We also show that the majority of RNA polymerase II transcription elongation complexes are physically excluded from mitotic chromosomes between late prophase and late telophase. Despite generalized transcription repression and stripping of RNA polymerase II complexes from DNA, arrested RNA polymerase II ternary complexes appear to remain on some gene regions during mitosis. The cyclic repression of transcription and ejection of RNA polymerase II transcription elongation complexes may help regulate the transcriptional events that control cell cycle progression and differentiation.

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NTP-driven translocation and regulation of downstream template opening by multi-subunit RNA polymerases

Biochemistry and Cell Biology ◽

10.1139/o05-059 ◽

2005 ◽

Vol 83 (4) ◽

pp. 486-496 ◽

Cited By ~ 28

Author(s):

Zachary F Burton ◽

Michael Feig ◽

Xue Q Gong ◽

Chunfen Zhang ◽

Yuri A Nedialkov ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Active Site ◽

Binding Sites ◽

Rna Synthesis ◽

Rna Polymerases ◽

Phosphoryl Transfer ◽

Elongation Complex ◽

Detailed Model ◽

Loop 2

Multi-subunit RNA polymerases bind nucleotide triphosphate (NTP) substrates in the pretranslocated state and carry the dNMPNTP base pair into the active site for phosphoryl transfer. NTP-driven translocation requires that NTP substrates enter the main-enzyme channel before loading into the active site. Based on this model, a new view of fidelity and efficiency of RNA synthesis is proposed. The model predicts that, during processive elongation, NTP-driven translocation is coupled to a protein conformational change that allows pyrophosphate release: coupling the end of one bond-addition cycle to substrate loading and translocation for the next. We present a detailed model of the RNA polymerase II elongation complex based on 2 low-affinity NTP binding sites located in the main-enzyme channel. This model posits that NTP substrates, elongation factors, and the conserved Rpb2 subunit fork loop 2 cooperate to regulate opening of the downstream transcription bubble.Key words: RNA polymerase, NTP-driven translocation, transcriptional fidelity, transcriptional efficiency, α-amanitin.

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Transient Reversal of RNA Polymerase II Active Site Closing Controls Fidelity of Transcription Elongation

Molecular Cell ◽

10.1016/j.molcel.2008.04.017 ◽

2008 ◽

Vol 30 (5) ◽

pp. 557-566 ◽

Cited By ~ 128

Author(s):

Maria L. Kireeva ◽

Yuri A. Nedialkov ◽

Gina H. Cremona ◽

Yuri A. Purtov ◽

Lucyna Lubkowska ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Active Site ◽

Transcription Elongation

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Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1520555113 ◽

2016 ◽

Vol 113 (15) ◽

pp. 4051-4056 ◽

Cited By ~ 54

Author(s):

Bin Liu ◽

Yuhong Zuo ◽

Thomas A. Steitz

Keyword(s):

Rna Polymerase ◽

Conformational Changes ◽

Active Site ◽

Transcription Initiation ◽

De Novo ◽

Addition Reaction ◽

Specific Interactions ◽

Promoter Recognition ◽

Nucleotide Addition ◽

Promoter Dna

In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3′-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E. coli transcription initiation complexes (TICs) containing the stress-responsive σS factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σS-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σS factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the −10 element. In addition, σS-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σS-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation.

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Ancient Transcription Factors in the News

mBio ◽

10.1128/mbio.01547-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 10

Author(s):

Irina Artsimovitch ◽

Stefan H. Knauer

Keyword(s):

Transcription Factors ◽

Nucleic Acids ◽

Rna Polymerase ◽

Rna Processing ◽

Binding Sites ◽

Rna Synthesis ◽

Mechanistic Studies ◽

Elongation Complex ◽

Content Type ◽

Domains Of Life

ABSTRACTIn every cell from bacteria to mammals, NusG-like proteins bind transcribing RNA polymerase to modulate the rate of nascent RNA synthesis and to coordinate it with numerous cotranscriptional processes that ultimately determine the transcript fate. Housekeeping NusG factors regulate expression of the bulk of the genome, whereas their highly specialized paralogs control just a few targets. InEscherichiacoli, NusG stimulates silencing of horizontally acquired genes, while its paralog RfaH counters NusG action by activating a subset of these genes. Acting alone or as part of regulatory complexes, NusG factors can promote uninterrupted RNA synthesis, bring about transcription pausing or premature termination, modulate RNA processing, and facilitate translation. Recent structural and mechanistic studies of NusG homologs from all domains of life reveal molecular details of multifaceted interactions that underpin their unexpectedly diverse regulatory roles. NusG proteins share conserved binding sites on RNA polymerase and many effects on the transcription elongation complex but differ in their mechanisms of recruitment, interactions with nucleic acids and secondary partners, and regulatory outcomes. Strikingly, some can alternate between autoinhibited and activated states that possess dramatically different secondary structures to achieve exquisite target specificity.

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NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble

eLife ◽

10.7554/elife.18096 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 36

Author(s):

Matti Turtola ◽

Georgiy A Belogurov

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Transcription Elongation ◽

Single Nucleotide ◽

E Coli ◽

Transcription Bubble ◽

Nucleotide Addition ◽

Nascent Rna ◽

Comprehensive Mapping

Universally conserved factors from NusG family bind at the upstream fork junction of transcription elongation complexes and modulate RNA synthesis in response to translation, processing, and folding of the nascent RNA. Escherichia coli NusG enhances transcription elongation in vitro by a poorly understood mechanism. Here we report that E. coli NusG slows Gre factor-stimulated cleavage of the nascent RNA, but does not measurably change the rates of single nucleotide addition and translocation by a non-paused RNA polymerase. We demonstrate that NusG slows RNA cleavage by inhibiting backtracking. This activity is abolished by mismatches in the upstream DNA and is independent of the gate and rudder loops, but is partially dependent on the lid loop. Our comprehensive mapping of the upstream fork junction by base analogue fluorescence and nucleic acids crosslinking suggests that NusG inhibits backtracking by stabilizing the minimal transcription bubble.

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Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011844 ◽

2020 ◽

Vol 295 (28) ◽

pp. 9583-9595

Author(s):

Aleksei Agapov ◽

Artem Ignatov ◽

Matti Turtola ◽

Georgiy Belogurov ◽

Daria Esyunina ◽

...

Keyword(s):

Rna Polymerase ◽

Stress Responses ◽

Active Site ◽

Rna Synthesis ◽

Conformational Dynamics ◽

Dna Lesions ◽

Trigger Loop ◽

Nucleotide Addition

DNA lesions can severely compromise transcription and block RNA synthesis by RNA polymerase (RNAP), leading to subsequent recruitment of DNA repair factors to the stalled transcription complex. Recent structural studies have uncovered molecular interactions of several DNA lesions within the transcription elongation complex. However, little is known about the role of key elements of the RNAP active site in translesion transcription. Here, using recombinantly expressed proteins, in vitro transcription, kinetic analyses, and in vivo cell viability assays, we report that point amino acid substitutions in the trigger loop, a flexible element of the active site involved in nucleotide addition, can stimulate translesion RNA synthesis by Escherichia coli RNAP without altering the fidelity of nucleotide incorporation. We show that these substitutions also decrease transcriptional pausing and strongly affect the nucleotide addition cycle of RNAP by increasing the rate of nucleotide addition but also decreasing the rate of translocation. The secondary channel factors DksA and GreA modulated translesion transcription by RNAP, depending on changes in the trigger loop structure. We observed that although the mutant RNAPs stimulate translesion synthesis, their expression is toxic in vivo, especially under stress conditions. We conclude that the efficiency of translesion transcription can be significantly modulated by mutations affecting the conformational dynamics of the active site of RNAP, with potential effects on cellular stress responses and survival.

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