Faculty Opinions recommendation of Antibacterial peptide microcin J25 inhibits transcription by binding within and obstructing the RNA polymerase 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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MSMEG_6292, a Mycobacterium smegmatis RNA polymerase secondary channel-binding protein: purification, crystallization and X-ray diffraction analysis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18009755 ◽

2018 ◽

Vol 74 (9) ◽

pp. 543-548

Author(s):

Abyson Joseph ◽

Valakunja Nagaraja ◽

Ramanathan Natesh

Keyword(s):

Rna Polymerase ◽

Mycobacterium Smegmatis ◽

Transcriptional Activity ◽

Molecular Replacement ◽

X Ray Diffraction ◽

Secondary Channel ◽

X Ray ◽

Cell Parameters ◽

Crystallization Method ◽

Better Than

The transcriptional activity of RNA polymerase (RNAP) is controlled by a diverse set of regulatory factors. A subset of these regulators modulate the activity of RNAP through its secondary channel. Gre factors reactivate stalled elongation complexes by enhancing the intrinsic cleavage activity of RNAP. In the present study, the protein MSMEG_6292, a Gre-factor homologue from Mycobacterium smegmatis, was expressed heterologously in Escherichia coli and purified using standard chromatographic techniques. The hanging-drop vapour-diffusion crystallization method yielded diffraction-quality crystals. The crystals belonged to the trigonal space group P3121 (or its enantiomorph P3221), with unit-cell parameters a = b = 83.15, c = 107.07 Å, α = β = 90, γ = 120°. The crystals diffracted to better than 3.0 Å resolution. Molecular-replacement attempts did not yield any phasing models; hence, platinum derivatization was carried out with K2PtCl4 and derivative data were collected to 3.4 Å resolution.

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Proton motive force dissipation precludes interaction of microcin J25 with RNA polymerase, but enhances reactive oxygen species overproduction

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/j.bbagen.2009.07.006 ◽

2009 ◽

Vol 1790 (10) ◽

pp. 1307-1313 ◽

Cited By ~ 4

Author(s):

Fernando G. Dupuy ◽

María V. Niklison Chirou ◽

Beatriz Fernández de Arcuri ◽

Carlos J. Minahk ◽

Roberto D. Morero

Keyword(s):

Reactive Oxygen Species ◽

Rna Polymerase ◽

Reactive Oxygen ◽

Proton Motive Force ◽

Motive Force ◽

Oxygen Species ◽

Microcin J25

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Regulation through the RNA Polymerase Secondary Channel

Journal of Biological Chemistry ◽

10.1074/jbc.c500405200 ◽

2005 ◽

Vol 281 (3) ◽

pp. 1309-1312 ◽

Cited By ~ 36

Author(s):

Jindrich Symersky ◽

Anna Perederina ◽

Marina N. Vassylyeva ◽

Vladimir Svetlov ◽

Irina Artsimovitch ◽

...

Keyword(s):

Rna Polymerase ◽

Secondary Channel

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An energy charge sensor for balancing RNA polymerase recycling and hibernation

10.21203/rs.3.rs-40617/v1 ◽

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.

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Escherichia coli DksA Binds to Free RNA Polymerase with Higher Affinity than to RNA Polymerase in an Open Complex

Journal of Bacteriology ◽

10.1128/jb.00621-09 ◽

2009 ◽

Vol 191 (18) ◽

pp. 5854-5858 ◽

Cited By ~ 27

Author(s):

Christopher W. Lennon ◽

Tamas Gaal ◽

Wilma Ross ◽

Richard L. Gourse

Keyword(s):

Escherichia Coli ◽

Transcription Factor ◽

Rna Polymerase ◽

Binding Affinity ◽

Quantitative Assay ◽

Secondary Channel ◽

Apparent Affinity ◽

Transcriptional Output

ABSTRACT The transcription factor DksA binds in the secondary channel of RNA polymerase (RNAP) and alters transcriptional output without interacting with DNA. Here we present a quantitative assay for measuring DksA binding affinity and illustrate its utility by determining the relative affinities of DksA for three different forms of RNAP. Whereas the apparent affinities of DksA for RNAP core and holoenzyme are the same, the apparent affinity of DksA for RNAP decreases almost 10-fold in an open complex. These results suggest that the conformation of RNAP present in an open complex is not optimal for DksA binding and that DNA directly or indirectly alters the interface between the two proteins.

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