Unknown,Transcriptomics,Genomics,Proteomics

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The omega subunit of the RNA polymerase core directs transcription efficiency in cyanobacteria

ABSTRACT: The catalytic core of the RNA polymerase of most eubacteria is composed of two M-NM-1 subunits and M-NM-2, M-NM-2M-bM-^@M-^Y and M-OM-^I subunits. In Escherichia coli, the M-OM-^I subunit (encoded by the rpoZ gene) has been suggested to assist M-NM-2M-bM-^@M-^Y during RNA polymerase core assembly. The function of the M-OM-^I subunit is particularly interesting in cyanobacteria because the cyanobacterial M-NM-2M-bM-^@M-^Y is split to N-terminal M-NM-3 and C-terminal M-NM-2M-bM-^@M-^Y subunits. The M-bM-^HM-^FrpoZ strain of the cyanobacterium Synechocystis sp. PCC 6803 grew well in standard conditions although the mutant cells showed low light-saturated photosynthetic activity, low Rubisco content and accumulated high quantities of protective carotenoids and M-NM-1-tocopherol. The M-bM-^HM-^FrpoZ strain contained 15% less of the primary M-OM-^C factor, SigA, than the control strain, and recruitment of SigA to the RNA polymerase core was inefficient in M-bM-^HM-^FrpoZ. Thus, a cyanobacterial RNA polymerase holoenzyme lacking the M-OM-^I subunit contains less frequently the primary M-OM-^C factor. A DNA microarray analysis revealed that this leads to specific down-regulation of highly expressed genes, like genes encoding subunits for Rubisco, ATP synthase, NADH-dehydrogenase and carbon concentrating mechanisms. On the contrary, many genes showing only low or moderate expression in the control strain were up-regulated in M-bM-^HM-^FrpoZ. A conserved -10 region was detected in promoters showing up or down-regulation in M-bM-^HM-^FrpoZ, but -35 regions of down-regulated genes completely differed from -35 regions of up-regulated genes. Cells from cyanobacteria Synechocystis sp. PCC 6803 named as control strain (CS) and RNA polymerase omega subunit inactivation strain, M-NM-^TrpoZ, were harvested (A730=1, 40 mL) directly from standard growth conditions (continuous illumination at the PPFD of 40 M-BM-5mol m-2s-1, 32M-BM-0C, ambient CO2). From three to four independent experiments were performed at each conditions.

ORGANISM(S): Synechocystis sp. PCC 6803

SUBMITTER: Taina Tyystjarvi

PROVIDER: E-GEOD-51647 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Adjustment of gene expression during acclimation to stress conditions, such as bright light, in the cyanobacterium Synechocystis sp. PCC 6803 depends on four group 2 M-OM-^C factors (SigB, SigC, SigD, SigE). A M-bM-^HM-^FsigCDE strain containing the stress responsive SigB as an only functional group 2 M-OM-^C factor appears twice as resistant to photoinhibition of photosystem II (PSII) as the control strain. Microarray analyzes of the M-bM-^HM-^FsigCDE strain indicated that 77 genes in standard conditions and 79 genes in high light were differently expressed compared to the control strain. Analysis of possible photoprotective mechanisms revealed that high carotenoid content and up-regulation of the photoprotective flavodiiron operon flv4-sll0218-flv2 protected PSII in M-bM-^HM-^FsigCDE, while up-regulation of pgr5-like, hlipB or isiA genes in the mutant strain did not offer particular protection against photoinhibition. The photoinhibition resistance was lost if M-bM-^HM-^FsigCDE was grown in high CO2 where carotenoid and Flv4-Sll0218-Flv2 contents were low. Additionally, photoinhibition resistance of the M-bM-^HM-^FrpoZ strain (lacking the omega subunit of RNA polymerase) with very high carotenoid but only low Flv4-Sll0218-Flv2 content supported the importance of carotenoids in PSII protection. Carotenoids likely protect mainly via quenching of singlet oxygen but efficient non-photochemical quenching in M-bM-^HM-^FsigCDE might offer some additional protection. Comparison of photoinhibition kinetics in control, M-bM-^HM-^FsigCDE and M-bM-^HM-^FrpoZ strains showed that protection by the flavodiiron operon was most efficient during the first minutes of high-light illumination. Cells from cyanobacteria Synechocystis sp. PCC 6803 named as control strain (CS) and group 2 sigma factor mutant M-NM-^TsigCDE (A730=1, 40 mL) were harvested directly from standard growth conditions (continuous illumination at the PPFD of 40 M-BM-5mol m-2s-1, 32M-BM-0C, ambient CO2) or after 30-min illumination a PPFD 750 M-BM-5mol m-2s-1. From three to four independent experiments were performed at each conditions.

Project description:Proper chromatin organization is essential for defining transcription units and maintaining genomic integrity in eukaryotes. Mutations affecting the chromatin structure can lead to increased cryptic transcription and genomic instability. In this study we found that deletion of the Schizosaccharomyces pombe Chd1-type chromatin remodelers, hrp1 and hrp3, causes strong, genome-wide accumulation of antisense transcripts, while the amount of coding mRNA transcripts is mostly unaffected. Nucleosome mapping revealed a specific role for Chd1-remodelers in the positioning of nucleosomes in gene coding regions. While the arrangement of nucleosomes in promoter regions was similar to WT, nucleosome organization within coding regions was remarkably irregular in hrp1M-bM-^HM-^Fhrp3M-bM-^HM-^F strain. We extended our analysis to other mutations associated with enhanced cryptic transcription activity, such as set2M-bM-^HM-^F, alp13M-bM-^HM-^F, and FACT complex subunit pob3M-bM-^HM-^F. While nucleosomes were severely depleted in the pob3M-bM-^HM-^F strain, nucleosome positioning was less affected. In sharp contrast, nucleosome organization in the alp13M-bM-^HM-^F and set2M-bM-^HM-^F strains was indistinguishable from WT. These data indicate multiple mechanisms in the repression of cryptic promoter activity in eukaryotic cells. Genome-wide profiling of H3K9/K14 acetylation Genome-wide expression analysis of either Alp13-, Set2-, Hrp3 or Hrp1 and Hrp3-deficient cells Genome-wide expression analysis of either Hrp1, Hrp3, or Hrp1 and Hrp3-deficient cells Nucleosome mapping experiments ChIP for the detection of the genome-wide acetylation profile of H3K9/K14 was performed for a wildtype strain and the deletion strains of Set2, Alp13 and the double knock-out of Hrp1 and Hrp3. Each experiment was performed twice in biological replicates All experiments were performed twice in biological replicates, except for the expression array of set2M-NM-^T. The replicates of hrp3M-NM-^T and hrp1M-NM-^Thrp3M-NM-^T were performed with a slightly different array design All experiments were performed twice in biological replicates. The replicates of hrp3M-NM-^T and hrp1M-NM-^Thrp3M-NM-^T were performed with a slightly different array design MNase treated sample were comparatively hybridized with genomic DNA of corresponding strain, WT, hrp1d hrp3d, pob3d in biological duplicates, set2d, alp13d, mit1d, hrp1d, hrp3d analysis was performed once

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The omega subunit of the RNA polymerase core directs transcription efficiency in cyanobacteria

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