Proteomics

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Crosslinking/mass spectrometry analysis of Bacillus subtilis RNAP-Delta-HelD complexes

ABSTRACT: Crosslinking mass spectrometry was applied to investigate protein contacts and dramatic structural rearrangements triggered by HelD binding to RNAP. Four complexes RNAPΔδΔHelD , RNAPΔδΔHelD-δ, RNAPΔδΔHelD-HelD and RNAPΔδΔHelD-δ-HelD were assembled using RNAP purified from B. subtilis ΔhelDΔropE strain (RNAPΔδΔHelD), and recombinant δ and HelD. The four complexes were crosslinked using a heterobifunctional, photoactivatable crosslinker sulfosuccinimidyl 4,4′‐azipentanoate (sulfo-SDA). Crosslinked protein complexes were analyzed by LC-MS/MS, and crosslinked residue pairs were identified in form of crosslinked peptides.

ORGANISM(S): Cellular Organisms

SUBMITTER: Juri Rappsilber 

PROVIDER: PXD019437 | JPOST Repository | Mon Oct 26 00:00:00 GMT 2020

REPOSITORIES: jPOST

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Dataset's files

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B200221_03_Lumos_ZC_CO_165_S3RNAP-SDA-10per.raw Raw
B200221_07_Lumos_ZC_CO_165_S1RNAP_Delta-SDA-10per.raw Raw
B200221_09_Lumos_ZC_CO_165_S4RNAP_HelD-SDA-10per.raw Raw
B200221_11_Lumos_ZC_CO_165_S2RNAP_Delta_HelD-SDA-10per.raw Raw
B200304_02_Lumos_ZC_CO_140_xBacillusRNAP-S3-SDA-SECf5.raw Raw
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Publications

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

Pei Hao-Hong HH   Hilal Tarek T   Chen Zhuo A ZA   Huang Yong-Heng YH   Gao Yuan Y   Said Nelly N   Loll Bernhard B   Rappsilber Juri J   Belogurov Georgiy A GA   Artsimovitch Irina I   Wahl Markus C MC  

Nature communications 20201218 1

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 a  ...[more]

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