Dataset Information

Proteins of anti-sigma70 and anti-RNAP immunoprecipitates in bacteria

ABSTRACT: As part of the project aimed at the interaction of RNA with RNA polymerase and primary sigma factor in bacteria we analyzed anti-σ70 antibody immunoprecipitates from Mycobacterium smegmatis stationary phase to identify proteins in σA complex in non-treated cells. We performed mass spectrometry analysis of anti-σ70 antibody and anti-RNAP immunoprecipitates from Corynebacterium glutamicum stationary phase to identify proteins present in σA and RNAP complexes.

INSTRUMENT(S): timsTOF SCP

ORGANISM(S): Mycobacterium Smegmatis (strain Atcc 700084 / Mc(2)155) Corynebacterium Glutamicum Atcc 13032

SUBMITTER: Petr Halada

LAB HEAD: Petr Novak

PROVIDER: PXD047705 | Pride | 2024-03-06

REPOSITORIES: Pride

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Publications

RIP-seq reveals RNAs that interact with RNA polymerase and primary sigma factors in bacteria.

Vaňková Hausnerová Viola V Shoman Mahmoud M Kumar Dilip D Schwarz Marek M Modrák Martin M Jirát Matějčková Jitka J Mikesková Eliška E Neva Silvia S Herrmannová Anna A Šiková Michaela M Halada Petr P Novotná Iva I Pajer Petr P Valášek Leoš Shivaya LS Převorovský Martin M Krásný Libor L Hnilicová Jarmila J

Nucleic acids research 20240501 8

Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S ...[more]

PMID: 38348908

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Project description:We developed native elongating transcript sequencing (NET-seq, Churchman and Weissman Nature 2011, PMID: 21248844) combined with RNase footprinting of nascent transcripts (RNET-seq) to visualize translocation dynamics and nascent transcript errors in paused RNA polymerases in E. coli. We employed RNET-seq to the wild-type (WT) E. coli strain and to an isogenic strain deficient in genes for GreA and GreB (ΔgreAB). Gre factors and their eukaryotic analog TFIIS rescue backtracked complexes of RNAP. Briefly, the cells were rapidly lysed via spheroplasting, and the transcribing RNAPs were released from the genomic DNA by digestion with DNase I. Any ribosomes involved in co-transcriptional translation were separated from RNAP by digestion with RNase A. All RNAPs including those associated with the fragmented double-stranded DNAs and their 5’-truncated nascent RNAs were immobilized on Ni2+-NTA beads through the hexa-histidine-tagged β’ subunit and then extensively washed with a high-salt buffer. The 5’ ends of the transcripts in ECs were trimmed with RNase T1/V1 to leave a minimal length of RNA protected by RNAP. The RNases were subsequently removed by further washing of the beads. Elution with imidazole generated ECs carrying ~6-30 nt long transcripts. The nascent RNAs isolated from the ΔgreAB strain were longer than those from the WT strain and peaked at 18 nt versus 16 nt suggesting an enrichment of backtracked ECs, which is expected to occur in the absence of Gre-dependent 3’ RNA cleavage. The RNET-seq method involves trimming of excess nascent RNA from the 5’ ends leaving only the nascent RNA that is protected by RNAP. A previous in vitro study showed that an RNAP forming an EC protects different lengths of the 3’-proximal transcript from trimming by RNases A and T1 depending on the EC translocation state. Post-translocated, pre-translocated, and backtracked complexes protect 14-nt, 15-nt and >15-nt segments of the RNA, respectively. Because the very 3’ end of the RNA is extruded to a narrow pore within front of the enzyme during backtracking, the extruded RNA remains inaccessible to RNases increasing in length as backtracking increases. Thus, paused RNAP in either the pre- and post-translocated states as well as in different backtracked distances were monitored over the entire genome. The unique properties of our RNET-seq approach provided an opportunity to dissect the core mechanisms of different types of pausing in living cells.

			Action	DRS
	CORGL_IgG.csv	Csv
	CORGL_IgG_A1_1_1843.d.zip	Other
	CORGL_RNAP.csv	Csv
	CORGL_RNAP_A1_1_1847.d.zip	Other
	CORGL_Sigma.csv	Csv