Genome-wide mapping of the distribution of CarD, RNAP Ï...A, and RNAP Î2 on the Mycobacterium smegmatis chromosome using chromatin immunoprecipitation sequencing

wide mapping of the distribution of CarD, RNAP σA, and RNAP β on the Mycobacterium smegmatis chromosome using chromatin immunoprecipitation sequencing. A , and RNAP β on the Mycobacterium smegmatis chromosome using chromatin immunoprecipitation sequencing a b s t r a c t a r t i c l e i n f o CarD is an essential mycobacterial protein that binds the RNA polymerase (RNAP) and affects the transcriptional profile of Mycobacterium smegmatis and Mycobacterium tuberculosis [6]. We predicted that CarD was directly regulating RNAP function but our prior experiments had not determined at what stage of transcription CarD was functioning and at which genes CarD interacted with the RNAP. To begin to address these open questions, we performed chromatin immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. The distribution of RNAP subunits β and σ A were also profiled. We expected that RNAP β would be present throughout transcribed regions and RNAP σ A would be predominantly enriched at promoters based on work in Escherichia coli [3], however this had yet to be determined in mycobacteria. The ChIP-seq analyses revealed that CarD was never present on the genome in the absence of RNAP, was primarily associated with promoter regions, and was highly correlated with the distribution of RNAP σ A. The colocalization of σ A 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. Here we describe in detail the data from the ChIP-seq experiments associated with the study published by Srivastava and colleagues in the Proceedings of the National Academy of Science in 2013 [5] as well as discuss the findings from this dataset in relation to both CarD and mycobacterial transcription as a whole. The ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) database, Specifications Sample and organism Genomic DNA from Mycobacterium smegmatis mc 2 155 derived strains Sequencer AB SOLiD 4 system high-throughput genome sequencer Data format Experimental factors In the M. smegmatis strain that was used, the carD gene had been deleted from the native chromosomal locus and the strain instead constitutively expressed a functional C-terminal HA tagged version of CarD. The exception was the control strain that expressed an untagged HA peptide and retained the carD gene at its endogenous locus. Experimental features All M. smegmatis …