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The DUF1013 protein TrcR tracks with RNA polymerase to control bacterial cell cycle and protection against antibiotics (RNA-seq dataset)

ABSTRACT: The regulatory roles of proteins associating with DNA-dependent RNA polymerase (RNAP) during transcription elongation are poorly characterized in bacteria. A forward genetic selection for Caulobacter crescentus cell cycle mutants revealed the uncharacterized DUF1013 protein (TrcR, transcriptional cell cycle regulator). TrcR associates with promoters and coding sequences (CDSs) and tracks with active RNAP. Loss of TrcR causes a cell division cycle and growth defect and an insufficiency in the essential cell cycle regulator CtrA. TrcR also protects cells from the quinolone antibiotic nalidixic acid that induces a multi-drug efflux pump and from the RNAP inhibitor rifampicin (Rif) that prevents transcription elongation. TrcR interacts biochemically and genetically with RNAP and no longer associates with chromatin when RNAP is inhibited with Rif. We show that these TrcR functions and its RNAP-dependent chromatin recruitment are conserved in symbiotic Sinorhizobium sp. and pathogenic Brucella sp., indicating that TrcR represents a new antibiotic target.

ORGANISM(S): Caulobacter vibrioides NA1000

PROVIDER: GSE148653 | GEO | 2021/02/03

REPOSITORIES: GEO

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Project description:CarD is an essential mycobacterial protein that we had previously shown to bind the RNA polymerase (RNAP) and affect the transcriptional profile of M. smegmatis and Mycobacterium tuberculosis. For this reason, we suspected that CarD was directly regulating transcriptional complexes but we did not know at what stage of CarD was functioning and at which genes CarD interacted with the RNAP. To determine in which stage of the transcription cycle (initiation, elongation, or termination) CarD acts, we used Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. Specific antibodies targeting core RNAPb, RNAPσ, or a hemagglutinin (HA) epitope fused to CarD (CarD-HA) were used to co-immunoprecipitate associated DNA. CarD-HA was immunoprecipitated from the M. smegmatis Mc2155 ΔcarD attB::tetcarD-HA strain and unfused HA was immunoprecipitated from the Mc2155 attB::pmsg431 strain with monoclonal antibodies specific for HA (Sigma). RNAP β and σ were immunoprecipitated from M. smegmatis ΔcarD attB::tetcarD-HA with monoclonal antibodies specific for these subunits (Neoclone, Madison, WI; 8RB13 for β, 2G10 for σ). Co-precipitated DNA was sequenced using a SOLiD sequencer (Life Technologies), which provided sufficient reads for 100-fold coverage of the genome. The number of sequence reads per base pair was normalized to the total number of reads and expressed as a log2 value. The reads per base pair from the HA-alone sample served as the background and was subtracted from the other datasets. We found that CarD was never present on the genome in the absence of RNAP. However, whereas RNAP core enzyme was found throughout transcribed regions of the genome, CarD was primarily associated with promoter regions and highly correlated with RNAPσ. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. The genome sequences associated with M. smegmatis CarD, RNAPb, and RNAPs were determined by ChIP-seq analysis. Samples were done in duplicate, except for RNAPs. And sequencing was performed using a SOLiD sequencer (Life Technologies).

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The DUF1013 protein TrcR tracks with RNA polymerase to control bacterial cell cycle and protection against antibiotics (RNA-seq dataset)

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