The torpedo effect in<i>Bacillus subtilis</i>:<scp>RN</scp>ase J1 resolves stalled transcription complexes

Šiková, Michaela; Wiedermannová, Jana; Převorovský, Martin; Barvı́k, Ivan; Sudzinová, Petra; Kofroňová, Olga; Benada, Oldřich; Šanderová, Hana; Condon, Ciarán; Krásný, Libor

doi:10.15252/embj.2019102500

Cited by 31 publications

(20 citation statements)

References 104 publications

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“…Concerning the potential RNase Y-based degradosome of the Gram-positive bacterium B. subtilis , the results were rather contradictory. RNase Y is, as expected, membrane associated, but RNase J1, which is one of its proposed interacting partners, does not follow the same distribution, being cytoplasmic ( 25 , 33 ). In contrast, in a recent work, a fraction of RNase J2 of Streptococcus mutans was found to localize at the membrane ( 34 ).…”

Section: Discussionsupporting

confidence: 72%

The RNase J-Based RNA Degradosome Is Compartmentalized in the Gastric Pathogen Helicobacter pylori

Tejada-Arranz

Galtier

Mortaji

et al. 2020

mBio

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Posttranscriptional regulation is a major level of gene expression control in any cell. In bacteria, multiprotein machines called RNA degradosomes are central for RNA processing and degradation, and some were reported to be compartmentalized inside these organelleless cells. The minimal RNA degradosome of the important gastric pathogen Helicobacter pylori is composed of the essential ribonuclease RNase J and RhpA, its sole DEAD box RNA helicase, and plays a major role in the regulation of mRNA decay and adaptation to gastric colonization. Here, the subcellular localization of the H. pylori RNA degradosome was investigated using cellular fractionation and both confocal and superresolution microscopy. We established that RNase J and RhpA are peripheral inner membrane proteins and that this association was mediated neither by ribosomes nor by RNA nor by the RNase Y membrane protein. In live H. pylori cells, we observed that fluorescent RNase J and RhpA protein fusions assemble into nonpolar foci. We identified factors that regulate the formation of these foci without affecting the degradosome membrane association. Flotillin, a bacterial membrane scaffolding protein, and free RNA promote focus formation in H. pylori. Finally, RNase J-GFP (RNase J-green fluorescent protein) molecules and foci in cells were quantified by three-dimensional (3D) single-molecule fluorescence localization microscopy. The number and size of the RNase J foci were found to be scaled with growth phase and cell volume as previously reported for eukaryotic ribonucleoprotein granules. In conclusion, we propose that membrane compartmentalization and the regulated clustering of RNase J-based degradosome hubs represent important levels of control of their activity and specificity. IMPORTANCE Helicobacter pylori is a bacterial pathogen that chronically colonizes the stomach of half of the human population worldwide. Infection by H. pylori can lead to the development of gastric pathologies such as ulcers and adenocarcinoma, which causes up to 800,000 deaths in the world each year. Persistent colonization by H. pylori relies on regulation of the expression of adaptation-related genes. One major level of such control is posttranscriptional regulation, which, in H. pylori, largely relies on a multiprotein molecular machine, an RNA degradosome, that we previously discovered. In this study, we established that the two protein partners of this machine are associated with the membrane of H. pylori. Using cutting-edge microscopy, we showed that these complexes assemble into hubs whose formation is regulated by free RNA and scaled with bacterial size and growth phase. Organelleless cellular compartmentalization of molecular machines into hubs emerges as an important regulatory level in bacteria.

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Section: Discussionsupporting

confidence: 72%

The RNase J-Based RNA Degradosome Is Compartmentalized in the Gastric Pathogen Helicobacter pylori

Tejada-Arranz

Galtier

Mortaji

et al. 2020

mBio

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“…Elongation complexes (ECs), containing a transcription bubble, were assembled with the Msm RNAP core as described before 22 . DNA and RNA oligonucleotides were purchased and are the same as in Table EV7 in 23 . The RNA (LK-pRNA) was monophosphorylated at the 5’ end by the manufacturer.…”

Section: Methodsmentioning

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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“…In bacteria, transcription and DNA replication occur concomitantly, making potentially damaging collisions of DNA replication forks with transcription complexes inevitable 1 – 5 . Transcription is highly sensitive to DNA damage, which causes the elongation complex (EC) to pause, and multiple redundant systems have evolved to ensure rapid removal of RNAP and/or the repair of damaged DNA 6 – 10 . This reduces the chance of replication forks colliding with stalled transcription complexes whilst also serving as an efficient system for maintaining genome integrity, especially within coding regions.…”

Section: Introductionmentioning

confidence: 99%

Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

et al. 2020

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In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.

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The torpedo effect inBacillus subtilis:RNase J1 resolves stalled transcription complexes

Cited by 31 publications

References 104 publications

The RNase J-Based RNA Degradosome Is Compartmentalized in the Gastric Pathogen Helicobacter pylori

The RNase J-Based RNA Degradosome Is Compartmentalized in the Gastric Pathogen Helicobacter pylori

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

Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

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