Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Mycobacterium smegmatis: CarD depletion (mgm1703) vs Control (mgm1701)

ABSTRACT: Transcriptional profiling of Mycobacterium smegmatis comparing the effects of wild type levels of CarD expression with CarD depletion. Transcriptional profile of the mgm1701 control strain was compared with that of mgm1703, a strain where CarD transcription is regulated by a Tet-On system. Both strains were grown in the absence of anhydrotetracline for 13 hours in nutrient rich broth to allow for depletion of CarD transcription in mgm1703. Two condition experiment, control (mgm1701) vs CarD depletion (mgm1703). 3 biological replicates of each strain.

ORGANISM(S): Mycobacterium smegmatis

SUBMITTER: Michael Glickman

PROVIDER: E-GEOD-15437 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

CarD is an essential regulator of rRNA transcription required for Mycobacterium tuberculosis persistence.

Stallings Christina L CL Stephanou Nicolas C NC Chu Linda L Hochschild Ann A Nickels Bryce E BE Glickman Michael S MS

Cell 20090701 1

Mycobacterium tuberculosis is arguably the world's most successful infectious agent because of its ability to control its own cell growth within the host. Bacterial growth rate is closely coupled to rRNA transcription, which in E. coli is regulated through DksA and (p)ppGpp. The mechanisms of rRNA transcriptional control in mycobacteria, which lack DksA, are undefined. Here we identify CarD as an essential mycobacterial protein that controls rRNA transcription. Loss of CarD is lethal for mycobac ...[more]

PMID: 19596241

Similar Datasets

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).

Dataset Information

Mycobacterium smegmatis: CarD depletion (mgm1703) vs Control (mgm1701)

Publications

CarD is an essential regulator of rRNA transcription required for Mycobacterium tuberculosis persistence.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets