Alternative transcription cycle for bacterial RNA polymerase

Harden, Timothy T.; Herlambang, Karina S.; Chamberlain, Mathew; Lalanne, Jean-Benoît; Wells, Christopher D.; Li, Gene-Wei; Landick, Robert; Hochschild, Ann; Kondev, Jané; Gelles, Jeff

doi:10.1038/s41467-019-14208-9

Cited by 37 publications

(73 citation statements)

References 65 publications

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“…To further delineate the contributions of δ and HelD to nucleic acid displacement, we conducted band shift assays, in which we first bound RNAP to nucleic acids and subsequently added δ and/or HelD. We first tested displacement of DNA with an artificial bubble, which when bound to RNAP mimics a situation ensuing after many intrinsic termination events [3][4][5]40 . HelD displaced about 25% of DNA from RNAP ΔδΔHelD , while δ led to about 80% displacement in the absence of HelD (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…At a terminator, bacterial EC is abruptly destabilized either by an oligo-U-tailed G/C-rich RNA hairpin or by the RNA translocase/helicase ρ 2 . However, RNAP can linger on DNA after RNA release 3 – 5 , roadblocking replisomes to trigger double-stranded DNA breaks 6 and giving rise to aberrant antisense transcripts 5 . RNAP can also form binary complexes with RNA 7 , 8 , either through de novo association with stable RNAs, such as tRNAs and 6S RNA 9 , 10 , or in the course of hairpin-induced termination 11 .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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“…Based on the structural and functional data we propose a role for Msm HelD in transcription (Figure 5). We envisage that upon transcription termination when RNAP fails to dissociate from nucleic acids 28 , or in the event of stalled elongation, Msm HelD first interacts with RNAP by its N-terminal domain, likely competing for binding to the secondary channel with GreA-like factors. This initial HelD binding induces changes in β-domain 2 and β’-jaw/shelf (Figure S8g), possibly leading to destabilisation of DNA in the primary channel.…”

Section: Discussionmentioning

confidence: 99%

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

Kouba

Kovaĺ

Sudzinová

et al. 2020

Preprint

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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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“…Colocalization Single Molecule Spectroscopy (CoSMoS) is a multi-wavelength single-molecule fluorescence method well suited for characterizing molecular mechanisms (21,22). We have previously used this approach to analyze the pathways of molecular complex formation and function in bacterial transcription and pre-mRNA splicing (22)(23)(24)(25)(26)(27)(28)(29). By fluorescently labeling the endogenous RNApII and Spt4/5 in nuclear extract, as well as the DNA template, with three differently colored dyes, we can monitor the arrival and departure of the individual protein molecules from the template DNA.…”

Section: Introductionmentioning

confidence: 99%

Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

Rosen¹,

Baek

Friedman³

et al. 2020

Preprint

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In eukaryotes, RNA polymerase II (RNApII) transcribes messenger RNA from template DNA. Decades of experiments have identified the proteins needed for transcription activation, initiation complex assembly, and productive elongation. However, the dynamics of recruitment of these proteins to transcription complexes, and of the transitions between these steps, are poorly understood. We used multi-wavelength single-molecule fluorescence microscopy to directly image and quantitate these dynamics in a budding yeast nuclear extract that reconstitutes activator-dependent transcription in vitro. A strong activator (Gal4-VP16) greatly stimulated reversible binding of individual RNApII molecules to template DNA, with no detectable involvement of RNApIIcontaining condensates. Binding of labeled elongation factor Spt4/5 to DNA typically followed RNApII binding, was NTP-dependent, and was correlated with association of mRNA-binding protein Hek2, demonstrating specificity of Spt4/5 binding to elongation complexes. Quantitative kinetic modeling shows that only a fraction of RNApII binding events are productive and implies a rate-limiting step, probably associated with recruitment of general transcription factors, needed to assemble a transcription-competent pre-initiation complex at the promoter. Spt4/5 association with transcription complexes was slowly reversible, with DNA-bound RNApII molecules sometimes binding and releasing Spt4/5 multiple times. The average Spt4/5 residence time was of similar magnitude to the time required to transcribe an average length yeast gene. These dynamics suggest that a single Spt4/5 molecule remains associated during a typical transcription event, yet can dissociate from RNApII to allow disassembly of abnormally long-lived (i.e., stalled) elongation complexes. Significance StatementThe synthesis of a eukaryotic messenger RNA molecule involves the association of RNA polymerase and dozens of accessory proteins on DNA. We used differently colored fluorescent dyes to tag DNA, RNA polymerase II, and the elongation factor Spt4/5 in yeast nuclear extract, and then observed the assembly and dynamics of individual molecules of the proteins with single DNA molecules by microscopy. The observations quantitatively define an overall pathway by which transcription complexes form and evolve in response to an activator protein. They suggest how molecular complex dynamics may be tuned to optimize efficient RNA production.

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Alternative transcription cycle for bacterial RNA polymerase

Cited by 37 publications

References 65 publications

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

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

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

Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

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