ChAP-chip analysis of RpoC binding profiles on the B. subtilis chromosome in the intact and the C-terminal truncated RNAP alpha subunit expressing cells.
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ABSTRACT: We constructed the B. subtilis strains in which the expression of the intact and the C-terminal truncated RNAP alpha subunit were induced by different stimulus, IPTG and xylose, under the control of Pspac and Pxyl promoters. Then, we performed ChAP-chip analysis to analyze the impact of M-^UM-^AM-A-CTD defect on genome-wide transcriptome profile.
INSTRUMENT(S): Affymetrix
ORGANISM(S): Bacillus subtilis subsp. subtilis str. 168
Project description:We constructed the B. subtilis strains in which the expression of the intact and the C-terminal truncated RNAP alpha subunit were induced by different stimulus, IPTG and xylose, under the control of Pspac and Pxyl promoters. Then, we performed ChAP-chip analysis to analyze the impact of alpha-CTD defect on genome-wide transcriptome profile.
Project description:Genome-wide binding profiles of AbrB and Abh was determined by use of ChAP (chromatin affinity purification)-chip method, modified method of ChIP-chip. Genetic modification was undertaken to abrB and abh genes to translationally fuse 2HC (coding sequence of twelve histidine and chitin binding domain) at the 3' ends to express AbrB-2HC and Abh-2HC proteins respectively.
Project description:To obtain an insight into the in vivo dynamics of RNA polymerase (RNAP) on the B. subtilis genome, we analyzed the distribution of ?A and ? subunits of RNAP and the NusA elongation factor on the genome in exponentially growing cells, using the ChAP (Chromatin Affinity Precipitation)-chip method. In contrast to E. coli RNAP, which often accumulates at the promoter-proximal region, B. subtilis RNAP is evenly distributed from the promoter to the coding sequences in the majority of genes. This finding suggests that B. subtilis RNAP recruited to the promoter promptly translocates away from the promoter to form the elongation complex. We detected RNAP accumulation in the promoter-proximal regions of some genes, most of which are attributed to transcription attenuation systems in the leader region. Our findings suggest that the differences in RNAP behavior during initiation and early elongation steps between E. coli and B. subtilis result in distinct strategies for post-initiation control of transcription. The E. coli mechanism involves trapping at the promoter and promoter-proximal pausing of RNAP in addition to transcription attenuation, whereas transcription attenuation in leader sequences is mainly employed in B. subtilis. Wild-type strain, Bacillus subtilis 168, was also used for RNA and genomic DNA extraction and analysis - RNA data was divided by genome DNA data to normalize (1) PCR bias and (2) copy number of RNA molecule per genome for multi-copy genome of exponentially growing bacteria.
Project description:Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis the nucleoid occlusion protein Noc protects chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP-on-Chip and bioinformatics, we have identified a consensus Noc-binding DNA sequence (NBS), and show that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro. NBSs inserted near the replication terminus bound Noc-YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS recruited Noc-YFP and conferred a severe Noc-dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division but that its activity is strictly localized by interaction with NBS sites in vivo. We propose that Noc not only serves as a spatial regulator of cell division to protect the nucleoid, but also a timing device with an important role in the co-ordination of chromosome segregation and cell division.
Project description:The direct roles of the NO-sensitive NsrR repressor and the ResD response regulator in transcriptional control in Bacillus subtilis
Project description:Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate intrinsic cleavage of the nascent transcript at the active site of RNAP. Biochemical and genetic studies to date have shown that E. coli Gre factors prevent transcriptional arrest during elongation and enhance transcription fidelity. Furthermore, Gre factors participate in stimulation of promoter escape and suppression of promoter-proximal pausing during beginning of RNA synthesis in E. coli. Although Gre factors are conserved in general bacteria, limited functional studies have been performed in bacteria other than E. coli. In this investigation, ChAP-chip analysis was conducted to visualize the distribution of B. subtilis GreA on the chromosome and determine the effects of GreA inactivation on core RNAP trafficking. Our data show that GreA is uniformly distributed in the transcribed region from the promoter to coding region with core RNAP, and its inactivation induces RNAP accumulation at many promoter or promoter-proximal regions. Based on these findings, we propose that GreA would constantly associate with core RNAP during transcriptional initiation and elongation, and resolves its stalling at promoter or promoter-proximal regions, thus contributing to the even distribution of RNAP along the promoter and coding regions in B. subtilis cells.
Project description:Cell lines were generated by transfecting NS0 myeloma cells with a single vector which contained the genes for the heavy and light chain of the antibody anti-CD38, and also the selectable marker glutamine synthetase. After two rounds of limiting dilution cloning, long-term continuous culture was performed on two cell lines (4H8 and 4G3) to assess the stability of recombinant antibody production during periods of extended culture. Microarray analysis was performed on RNA samples extracted during log phase of growth at the start of long-term culture and after approximately one month.
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.