ChIP-chip of RNA Polymerase (RNAP) and RpoD in Salmonella enterica serovar Typhimurium strain SL1344
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ABSTRACT: ChIP-on-chip analysis of RNAP and RpoD binding to the Salmonella enterica serovar Typhimurium chromosome demonstrated a high degree of overlap between RNAP and RpoD binding and provided us with important insights into the global distribution of these factors. Furthermore this data was correlated with information on the location of 1873 transcription start sites identified by RNA-Seq technology, thereby providing a detailed transcriptional map of Salmonella Typhimurium. Analysis of RNAP, RNAP-Rifampicin and and RpoD binding in Luria Broth (LB)
Project description:We applied a ChIP-chip approach to elucidate the binding profiles of RNAP and RpoD experimentally under different growth conditions. This technique localizes DNA fragments within DNA-protein complexes enriched by chromatin immunoprecipitation using high-density oligonucleotide tilling arrays. A 21 ChIP-chip study using immunoprecipitated DNA (IP-DNA) from three culture conditions for RNAP and four culture conditions for RpoD. The high-density oligonucleotide tiling arrays used consisted of 381,174 oligonucleotide probes spaced 20 bp apart (30-bp overlap between two probes) across the G. sulfurreducens genome (NimbleGen). Experiments were conducted as three bioliogical replicates (different cultures).
Project description:The LysR family transcription factor LeuO is believed to antagonize the global repressor H-NS. ChIP-on-chip analysis of LeuO, H-NS and RNAP binding to the Salmonella enterica serovar Typhimurium chromosome demonstrated a high degree of overlap between LeuO and H-NS regulated genes. Furthermore LeuO binding was associated with RNA polymerase recruitment, indicating a role for LeuO in activating transcription. Analysis of LeuO, H-NS and RNA Polymerase binding in Low-phosphate media (LPM)
Project description:This SuperSeries is composed of the following subset Series: GSE22497: Transcriptome analysis of Geobacter sulfurreducens under multiple growth conditions GSE22503: ChIP-chip of Geobacter sulfurreducens PCA with antibody against RNAP and RpoD under various conditions GSE22511: Genome-wide transcription start site determination of Geobacter sulfurreducens under multiple growth conditions Refer to individual Series
Project description:Genome-wide identification of RNA polymerase (RNAP) binding sites were performed in Klebsiella pneumoniae MGH 78578 (KP). Anti-RNAP is used to capture the RNAP in KP. ChIP-chip was performed on tiling array specifically made for KP. Comparison ChIP by anti-RNAP antibody vs ChIP by normal mouse IgG (control, mock IP)
Project description:The global regulator H-NS represses transcription in gram negative bacteria. Sfh is a homologue of H-NS and is encoded by plasmid pSfR27. Sfh provides a 'stealth' function that allows pSfR27 to be transmitted to a new host without disrupting the competitive fitness of the new host We used ChIP-on-chip to profile Sfh (3xFLAG-tagged) and H-NS binding sites in Salmonella Typhimurium strain SL1344 and found that Sfh provides its 'stealth' function by targeting a sub-set of H-NS bound genes that display reduced levels of H-NS occupancy with the SL1344 chromosome upon acquisition of plasmid pSfR27 Identification of Sfh binding sites in strains SL1344 (pSfR27) and SL1344 hns (pSfR27) as well as identification of H-NS binding sites in strains SL1344, SL1344 (pSfR27), and SL1344 (pSfR27) sfh
Project description:We performed Chromatin Immunoprecipitation (ChIP) and microarray hybridization analysis of CspC binding in Salmonella Typhimurium strain SL1344 which has been genetically engineered to express a 3xFLAG tagged CspC protein. Chromatin samples were prepared from SL1344 CspC 3xFLAG grown to exponential phase (OD600nm = 0.2). CspC FLAG ChIP and mock normal mouse IgG ChIP reactions were carried out. The purified ChIP DNA samples were hybridized to SL1344 tiling microarrays.
Project description:The in vivo trafficking patterns on DNA by the bacterial regulators of transcript elongation Sigma70, Rho, NusA, and NusG and the explanation for high promoter-proximal levels or peaks of RNA polymerase (RNAP) are unknown. Genome-wide ChIP-chip on E. coli revealed distinct association patterns of regulators as RNAP transcribes away from promoters (Rho first, then NusA, and then NusG). However, the interactions of elongating complexes with these regulators, including a weak interaction with Sigma70, did not differ significantly among most transcription units. A modest variation of NusG signal among genes reflected increased NusG interaction as transcription progresses, rather than functional specialization of elongating complexes. Promoter-proximal RNAP peaks were offset from Sigma70 peaks in the direction of transcription and co-occurred with NusA and Rho peaks, suggesting that the RNAP peaks reflected elongating, rather than initiating, complexes. However, inhibition of Rho did not increase RNAP levels within genes downstream of the RNAP peaks, suggesting the peaks are caused by a mechanism other than simple Rho-dependent attenuation. Chromatin immunoprecipitation (ChIP) experiments were performed using antibodies against RNA polymerase (Beta' subunit), Sigma70, NusA, NusG, or Rho. Differentially labeled ChIP DNA and genomic DNA were competitively hybridized to an E. coli K-12 MG1655 tiling array with overlapping probes at ~24bp spacing across the entire genome. The series contains 17 total datasets.
Project description:In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise, and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrackblocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo. Chromatin immunoprecipitation (ChIP) experiments were performed by using antibodies against RNA polymerase b subunit in wild-type and DdksA cells treated with 0.5mg/ml serine hydroxamate (SHX) or untreated. DksA and s70 enrichments were compared to RNAP enrichment by ChIP experiments using antibodies against s70 and DksA in wild-type cells (also in DdksA cells as a negative control for DksA ChIP-chip). Differentially labeled ChIP DNA and genomic DNA were competitively hybridized to an E. coli K-12 MG1655 tiling array with overlapping probes at ~12bp spacing across the entire genome. The series contains 19 datasets.
Project description:The transcription termination factor Rho is a global regulator of RNA polymerase (RNAP). Although individual Rho-dependent terminators have been studied extensively, less is known about the sites of RNAP regulation by Rho on a genome-wide scale. Using chromatin immunoprecipitation and microarrays (ChIP-chip), we examined changes in the distribution of Escherichia coli RNAP in response to the Rho-specific inhibitor bicyclomycin (BCM). We found ~200 Rho-terminated loci that were divided evenly into two classes: intergenic (at the ends of genes) and intragenic (within genes). The intergenic class contained noncoding RNAs such as small RNAs (sRNAs) and transfer RNAs (tRNAs), establishing a previously unappreciated role of Rho in termination of stable RNA synthesis. The intragenic class of terminators included a novel set of short antisense transcripts, as judged by a shift in the distribution of RNAP in BCM-treated cells that was opposite to the direction of the corresponding gene. These Rho-terminated antisense transcripts point to a novel role of noncoding transcription in E. coli gene regulation that may resemble the ubiquitous noncoding transcription recently found to play myriad roles in eukaryotic gene regulation. Chromatin immunoprecipitation (ChIP) experiments were performed using antibodies against RNA polymerase (Beta or Beta' subunit) in cells treated with 20ug/ml bicyclomycin or untreated cells. Differentially labeled ChIP DNA and genomic DNA were competitively hybridized to an E. coli K-12 MG1655 tiling array with overlapping probes at ~12bp spacing across the entire genome. The series contains 4 datasets.
Project description:This SuperSeries is composed of the following subset Series: GSE19230: The effect of sfh deletion on Salmonella Typhimurium gene expression GSE19231: Identification of Sfh and H-NS binding sites in the Salmonella Typhimurium genome Refer to individual Series