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The Role of α-CTD in the Genome-Wide Transcriptional Regulation of the Bacillus subtilis Cells.

Murayama S, Ishikawa S, Chumsakul O, Ogasawara N, Oshima T - PLoS ONE (2015)

Bottom Line: Transcriptomic and ChAP-chip analyses revealed that α-CTD deficiency reduced the transcription and RNAP binding of genes related to the utilization of secondary carbon sources, transition state responses, and ribosome synthesis.In E. coli, it is known that α-CTD also contributes to the expression of genes related to the utilization of secondary carbon sources and ribosome synthesis.Our results suggest that the biological importance of α-CTD is conserved in B. subtilis and E. coli, but that its specific roles have diversified between these two bacteria.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan.

ABSTRACT
The amino acid sequence of the RNA polymerase (RNAP) α-subunit is well conserved throughout the Eubacteria. Its C-terminal domain (α-CTD) is important for the transcriptional regulation of specific promoters in both Escherichia coli and Bacillus subtilis, through interactions with transcription factors and/or a DNA element called the "UP element". However, there is only limited information regarding the α-CTD regulated genes in B. subtilis and the importance of this subunit in the transcriptional regulation of B. subtilis. Here, we established strains and the growth conditions in which the α-subunit of RNAP was replaced with a C-terminally truncated version. Transcriptomic and ChAP-chip analyses revealed that α-CTD deficiency reduced the transcription and RNAP binding of genes related to the utilization of secondary carbon sources, transition state responses, and ribosome synthesis. In E. coli, it is known that α-CTD also contributes to the expression of genes related to the utilization of secondary carbon sources and ribosome synthesis. Our results suggest that the biological importance of α-CTD is conserved in B. subtilis and E. coli, but that its specific roles have diversified between these two bacteria.

No MeSH data available.


Related in: MedlinePlus

RNAP binding effects following the replacement of RpoAint with RpoAdel in the RNAP complex.(A) Typical RNAP binding profiles obtained from rpoAint- and rpoAdel-expressing cells; rpsU and yqeY are indicated, with the RNAP binding signal of each probe mapped to the corresponding position in the B. subtilis chromosome. The binding intensity (shown by vertical bars) was determined as the relative ratio of the signal intensities obtained for the hybridization of labeled DNA fragments prepared from the affinity purification with RpoC or RpoA (ChAP DNA) versus whole cell extract (control DNA) fractions in each experiment. The RNAP binding intensities determined by affinity purification with RpoC as bait are shown in lanes 1 and 2. The RNAP binding intensities determined by affinity purification with RpoA as bait are shown in lanes 3 and 4. The RNAP binding intensities in the rpoAint-expressing cells are shown in lanes 1 (SMS08) and 3 (SMS18), and those in rpoAdel-expressing cells are indicated in lanes 2 (SMS09) and 4 (SMS19). The arrangement of genes in the presented chromosomal region is indicated by thick arrows at the top of the figure. The RNAP binding profiles obtained from one (Exp. 1) of duplicate experiments are shown as representative. (B) Binding to the mtl operon is shown as a typical example of the reduced RNAP binding observed in rpoAdel-expressing cells. (C) Binding to the srf operon is shown as an example of the unique RNAP binding profile observed in rpoAdel-expressing cells, in which reductions were seen in the protein coding regions but not in the promoter proximal regions.
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pone.0131588.g005: RNAP binding effects following the replacement of RpoAint with RpoAdel in the RNAP complex.(A) Typical RNAP binding profiles obtained from rpoAint- and rpoAdel-expressing cells; rpsU and yqeY are indicated, with the RNAP binding signal of each probe mapped to the corresponding position in the B. subtilis chromosome. The binding intensity (shown by vertical bars) was determined as the relative ratio of the signal intensities obtained for the hybridization of labeled DNA fragments prepared from the affinity purification with RpoC or RpoA (ChAP DNA) versus whole cell extract (control DNA) fractions in each experiment. The RNAP binding intensities determined by affinity purification with RpoC as bait are shown in lanes 1 and 2. The RNAP binding intensities determined by affinity purification with RpoA as bait are shown in lanes 3 and 4. The RNAP binding intensities in the rpoAint-expressing cells are shown in lanes 1 (SMS08) and 3 (SMS18), and those in rpoAdel-expressing cells are indicated in lanes 2 (SMS09) and 4 (SMS19). The arrangement of genes in the presented chromosomal region is indicated by thick arrows at the top of the figure. The RNAP binding profiles obtained from one (Exp. 1) of duplicate experiments are shown as representative. (B) Binding to the mtl operon is shown as a typical example of the reduced RNAP binding observed in rpoAdel-expressing cells. (C) Binding to the srf operon is shown as an example of the unique RNAP binding profile observed in rpoAdel-expressing cells, in which reductions were seen in the protein coding regions but not in the promoter proximal regions.

Mentions: To examine the effects of α-CTD deficiency on RNAP binding, we assessed the RNAP binding profiles of the genes that showed highly reduced RNAP binding in rpoAdel cells upon visual inspection of the RNAP (especially RpoC-His) distributions. The typical distributions of RNAP (revealed by using RpoC-His or RpoA-His) on the B. subtilis chromosome are shown in Fig 5A, and the binding profiles of RNAP throughout the chromosome of B. subtilis (as assessed in duplicate experiments) are presented in S10 Fig.


The Role of α-CTD in the Genome-Wide Transcriptional Regulation of the Bacillus subtilis Cells.

Murayama S, Ishikawa S, Chumsakul O, Ogasawara N, Oshima T - PLoS ONE (2015)

RNAP binding effects following the replacement of RpoAint with RpoAdel in the RNAP complex.(A) Typical RNAP binding profiles obtained from rpoAint- and rpoAdel-expressing cells; rpsU and yqeY are indicated, with the RNAP binding signal of each probe mapped to the corresponding position in the B. subtilis chromosome. The binding intensity (shown by vertical bars) was determined as the relative ratio of the signal intensities obtained for the hybridization of labeled DNA fragments prepared from the affinity purification with RpoC or RpoA (ChAP DNA) versus whole cell extract (control DNA) fractions in each experiment. The RNAP binding intensities determined by affinity purification with RpoC as bait are shown in lanes 1 and 2. The RNAP binding intensities determined by affinity purification with RpoA as bait are shown in lanes 3 and 4. The RNAP binding intensities in the rpoAint-expressing cells are shown in lanes 1 (SMS08) and 3 (SMS18), and those in rpoAdel-expressing cells are indicated in lanes 2 (SMS09) and 4 (SMS19). The arrangement of genes in the presented chromosomal region is indicated by thick arrows at the top of the figure. The RNAP binding profiles obtained from one (Exp. 1) of duplicate experiments are shown as representative. (B) Binding to the mtl operon is shown as a typical example of the reduced RNAP binding observed in rpoAdel-expressing cells. (C) Binding to the srf operon is shown as an example of the unique RNAP binding profile observed in rpoAdel-expressing cells, in which reductions were seen in the protein coding regions but not in the promoter proximal regions.
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pone.0131588.g005: RNAP binding effects following the replacement of RpoAint with RpoAdel in the RNAP complex.(A) Typical RNAP binding profiles obtained from rpoAint- and rpoAdel-expressing cells; rpsU and yqeY are indicated, with the RNAP binding signal of each probe mapped to the corresponding position in the B. subtilis chromosome. The binding intensity (shown by vertical bars) was determined as the relative ratio of the signal intensities obtained for the hybridization of labeled DNA fragments prepared from the affinity purification with RpoC or RpoA (ChAP DNA) versus whole cell extract (control DNA) fractions in each experiment. The RNAP binding intensities determined by affinity purification with RpoC as bait are shown in lanes 1 and 2. The RNAP binding intensities determined by affinity purification with RpoA as bait are shown in lanes 3 and 4. The RNAP binding intensities in the rpoAint-expressing cells are shown in lanes 1 (SMS08) and 3 (SMS18), and those in rpoAdel-expressing cells are indicated in lanes 2 (SMS09) and 4 (SMS19). The arrangement of genes in the presented chromosomal region is indicated by thick arrows at the top of the figure. The RNAP binding profiles obtained from one (Exp. 1) of duplicate experiments are shown as representative. (B) Binding to the mtl operon is shown as a typical example of the reduced RNAP binding observed in rpoAdel-expressing cells. (C) Binding to the srf operon is shown as an example of the unique RNAP binding profile observed in rpoAdel-expressing cells, in which reductions were seen in the protein coding regions but not in the promoter proximal regions.
Mentions: To examine the effects of α-CTD deficiency on RNAP binding, we assessed the RNAP binding profiles of the genes that showed highly reduced RNAP binding in rpoAdel cells upon visual inspection of the RNAP (especially RpoC-His) distributions. The typical distributions of RNAP (revealed by using RpoC-His or RpoA-His) on the B. subtilis chromosome are shown in Fig 5A, and the binding profiles of RNAP throughout the chromosome of B. subtilis (as assessed in duplicate experiments) are presented in S10 Fig.

Bottom Line: Transcriptomic and ChAP-chip analyses revealed that α-CTD deficiency reduced the transcription and RNAP binding of genes related to the utilization of secondary carbon sources, transition state responses, and ribosome synthesis.In E. coli, it is known that α-CTD also contributes to the expression of genes related to the utilization of secondary carbon sources and ribosome synthesis.Our results suggest that the biological importance of α-CTD is conserved in B. subtilis and E. coli, but that its specific roles have diversified between these two bacteria.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan.

ABSTRACT
The amino acid sequence of the RNA polymerase (RNAP) α-subunit is well conserved throughout the Eubacteria. Its C-terminal domain (α-CTD) is important for the transcriptional regulation of specific promoters in both Escherichia coli and Bacillus subtilis, through interactions with transcription factors and/or a DNA element called the "UP element". However, there is only limited information regarding the α-CTD regulated genes in B. subtilis and the importance of this subunit in the transcriptional regulation of B. subtilis. Here, we established strains and the growth conditions in which the α-subunit of RNAP was replaced with a C-terminally truncated version. Transcriptomic and ChAP-chip analyses revealed that α-CTD deficiency reduced the transcription and RNAP binding of genes related to the utilization of secondary carbon sources, transition state responses, and ribosome synthesis. In E. coli, it is known that α-CTD also contributes to the expression of genes related to the utilization of secondary carbon sources and ribosome synthesis. Our results suggest that the biological importance of α-CTD is conserved in B. subtilis and E. coli, but that its specific roles have diversified between these two bacteria.

No MeSH data available.


Related in: MedlinePlus