In vivo organization of bacterial transcription initiation complexes at a genome-scale [TSS-seq]
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ABSTRACT:
ORGANISM(S): Escherichia coli str. K-12 substr. MG1655
PROVIDER: GSE54406 | GEO | 2021/01/13
REPOSITORIES: GEO
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Project description:RNA polymerase (RNAP) is essential for the transcription of genetic information encoded in genomes in all three domains of life. To determine the precise organization of bacterial transcription initiation complexes (TIC) in vivo, we exploited high-throughput sequencing to the DNA obtained from exonuclease-treated immunoprecipitates of the TIC. It reveals that sigma (σ) factor is mostly engaged in RNAP holoenzyme up to 13-14 nucleotides from transcription start site during abortive initiation.
2021-01-13 | GSE54405 | GEO
Project description:RNA polymerase (RNAP) is essential for the transcription of genetic information encoded in genomes in all three domains of life. To determine the precise organization of bacterial transcription initiation complexes (TIC) in vivo, we exploited high-throughput sequencing to the DNA obtained from exonuclease-treated immunoprecipitates of the TIC. It reveals that sigma (σ) factor is mostly engaged in RNAP holoenzyme up to 13-14 nucleotides from transcription start site during abortive initiation.
2021-01-13 | GSE54404 | GEO
Project description:RNA polymerase (RNAP) is essential for the transcription of genetic information encoded in genomes in all three domains of life. To determine the precise organization of bacterial transcription initiation complexes (TIC) in vivo, we exploited high-throughput sequencing to the DNA obtained from exonuclease-treated immunoprecipitates of the TIC. It reveals that sigma (σ) factor is mostly engaged in RNAP holoenzyme up to 13-14 nucleotides from transcription start site during abortive initiation.
2021-01-13 | GSE54403 | GEO
Project description:In eubacteria, replacement of one σ factor in the RNA polymerase (RNAP) holoenzyme by another one changes the transcription pattern. Cyanobacteria are eubacteria characterized by oxygenic photosynthesis and they typically encode numerous group 2 σ factors that closely resemble the essential primary σ factor. A mutant strain of the model cyanobacterium Synechocystis sp. PCC 6803 without functional group 2 σ factors (named as ΔsigBCDE) could not acclimate to heat, high salt, or bright light stress but in standard conditions ΔsigBCDE grew only 9% slower than the control strain. One-fifth of the genes in ΔsigBCDE were differently expressed compared to the control strain in standard growth conditions and several physiological changes in photosynthesis, and pigment and lipid compositions were detected. To directly analyze the σ factor content of RNAP holoenzyme in vivo, a His-tag was added to the γ subunit of RNAP in Synechocystis and RNAPs were collected. The results revealed that all group 2 σ factors were recruited by RNAP in standard conditions, but recruitment of SigB and SigC increased in heat stress, SigD in bright light, SigE in darkness and SigB, SigC and SigE in high salt, explaining the poor acclimation of ΔsigBCDE to these stress conditions.
2015-09-15 | GSE69981 | GEO
Project description:In eubacteria, replacement of one σ factor in the RNA polymerase (RNAP) holoenzyme by another one changes the transcription pattern. Cyanobacteria are eubacteria characterized by oxygenic photosynthesis and they typically encode numerous group 2 σ factors that closely resemble the essential primary σ factor. A mutant strain of the model cyanobacterium Synechocystis sp. PCC 6803 without functional group 2 σ factors (named as ΔsigBCDE) could not acclimate to heat, high salt, or bright light stress but in standard conditions ΔsigBCDE grew only 9% slower than the control strain. One-fifth of the genes in ΔsigBCDE were differently expressed compared to the control strain in standard growth conditions and several physiological changes in photosynthesis, and pigment and lipid compositions were detected. To directly analyze the σ factor content of RNAP holoenzyme in vivo, a His-tag was added to the γ subunit of RNAP in Synechocystis and RNAPs were collected. The results revealed that all group 2 σ factors were recruited by RNAP in standard conditions, but recruitment of SigB and SigC increased in heat stress, SigD in bright light, SigE in darkness and SigB, SigC and SigE in high salt, explaining the poor acclimation of ΔsigBCDE to these stress conditions. Cells from cyanobacteria Synechocystis sp. PCC 6803 named as control strain (CS) and a mutant strain without any functional group 2 sigma factors, ΔsigBCDE, were harvested (A730=1, 40 mL) directly from standard growth conditions (continuous illumination at the PPFD of 40 µmol m-2s-1, 32°C, ambient CO2). From three to four independent experiments were performed at each conditions.
2015-09-15 | E-GEOD-69981 | biostudies-arrayexpress
Project description:As part of the project aimed at the interaction of RNA with RNA polymerase and primary sigma factor in bacteria we analyzed anti-σ70 antibody immunoprecipitates from Mycobacterium smegmatis stationary phase to identify proteins in σA complex in non-treated cells. We performed mass spectrometry analysis of anti-σ70 antibody and anti-RNAP immunoprecipitates from Corynebacterium glutamicum stationary phase to identify proteins present in σA and RNAP complexes.
2024-03-06 | PXD047705 | Pride
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.
2013-08-01 | GSE48164 | GEO
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).
2013-08-01 | E-GEOD-48164 | biostudies-arrayexpress
Project description:Gene expression in E. coli is controlled by well-established mechanisms that activate or repress transcription. Here, we identify CedA as an unconventional transcription factor specifically associated with the RNA polymerase (RNAP) σ70 holoenzyme. Structural and biochemical analysis of CedA bound to RNAP reveal that it bridges distant domains of β and σ70 subunits to stabilize an open-promoter complex. Remarkably, CedA does so without contacting DNA. We further show that cedA is strongly induced in response to amino acid starvation, oxidative stress, and aminoglycosides. CedA provides a basal level of tolerance to these clinically relevant antibiotics, as well as to rifampicin and peroxide. Finally, we show that CedA modulates transcription of hundreds of bacterial genes, which explains its pleotropic effect on cell physiology and pathogenesis.
2023-09-17 | PXD039446 | Pride
Project description:When performed at single bp resolution, the genome-wide location, occupancy level, and structural organization of DNA binding proteins provides mechanistic insights into genome regulation. Here we use ChIP-exo to provide the first high resolution view of the epigenomic organization of the E. coli transcription machinery and nucleoid structural proteins when cells are growing exponentially and upon rapid reprogramming (acute heat shock). We suggest that indirect readout of DNA shape at the flanks of cognate motifs provide major contributions to site specificity at promoter positions -35/-24 and -10/-12. We examined the site specificity of three sigma factors (RpoD/70, RpoH/32, and RpoN/54), RNA polymerase (RNAP or RpoA, B, C) and two nucleoid proteins (Fis and IHF). Our results confirm and refine reports that RpoD binds most annotated promoters, whereas RpoH and RpoN bind a much smaller subset, each through their cognate motifs. However, only upon heat shock does RpoH becomes active for RNAP recruitment. In contrast, upon heat shock RpoD remains active at its cognate promoters (including at heat shock genes), whereas RpoN remains inactive at its cognate promoters. RpoN binds ~1,000 non-annotated RpoN motifs, which may reflect a large number of condition-specific transcription units. Occupancy patterns of sigma factors and RNAP suggest a common promoter recruitment mechanism that differs from the long-standing views that sigma and RNAP are co-recruited as a complex, and also simultaneously dissociate from promoters. Our findings suggest that sigma factors are recruited and/or maintained at most promoters via an RNAP-independent mechanism. When RNAP arrives, it dwells for a relatively short time before clearing the promoter, leaving sigma behind. Taken together these findings add new dimensions to how sigma factors achieve promoter specificity through DNA sequence and shape and redefine mechanistic steps in regulated promoter assembly in E. coli.
2022-02-21 | GSE189206 | GEO