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Rapid changes in gene expression: DNA determinants of promoter regulation by the concentration of the transcription initiating NTP in Bacillus subtilis.

Sojka L, Kouba T, Barvík I, Sanderová H, Maderová Z, Jonák J, Krásny L - Nucleic Acids Res. (2011)

Bottom Line: An important small molecule effector is the initiating nucleoside triphosphate (iNTP).At some promoters, an increasing iNTP concentration stimulates promoter activity, while a decreasing concentration has the opposite effect.Overall, it seems that various sequence combinations can result in promoter regulation by [iNTP] in B. subtilis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics of Bacteria, Institute of Microbiology, Academy of Sciences of the Czech Republic.

ABSTRACT
In bacteria, rapid changes in gene expression can be achieved by affecting the activity of RNA polymerase with small molecule effectors during transcription initiation. An important small molecule effector is the initiating nucleoside triphosphate (iNTP). At some promoters, an increasing iNTP concentration stimulates promoter activity, while a decreasing concentration has the opposite effect. Ribosomal RNA (rRNA) promoters from Gram-positive Bacillus subtilis are regulated by the concentration of their iNTP. Yet, the sequences of these promoters do not emulate the sequence characteristics of [iNTP]-regulated rRNA promoters of Gram-negative Escherichia coli. Here, we identified the 3'-promoter region, corresponding to the transcription bubble, as key for B. subtilis rRNA promoter regulation via the concentration of the iNTP. Within this region, the conserved -5T (3 bp downstream from the -10 hexamer) is required for this regulation. Moreover, we identified a second class of [iNTP]-regulated promoters in B. subtilis where the sequence determinants are not limited to the transcription bubble region. Overall, it seems that various sequence combinations can result in promoter regulation by [iNTP] in B. subtilis. Finally, this study demonstrates how the same type of regulation can be achieved with strikingly different promoter sequences in phylogenetically distant species.

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Related in: MedlinePlus

Changes in activity of selected B. subtilis promoters during outgrowth from stationary phase. Cells were grown in rich MOPS 20 amino acids medium until 3 h into stationary phase (time ‘0’). Subsequently, cells were diluted into fresh medium and RNA was extracted at the indicated time points. RNA transcribed from the tested promoter was quantitated and used as a measure of the promoter’s activity (‘Materials and Methods’ section). To simplify comparison of the promoters in terms of the proportional increase in their activity, the activities of the promoters were normalized to 1 at time 0. The actual relative activities of the promoters normalized to rrnB P2 (set as 1) at time 0 were as follows: rrnB P2 was 1, Pveg was 1.26 and rrnB P1 was 0.02. Thus, the activity of rrnB P1 was most repressed at time 0, allowing for the subsequent large increase in activity. Strains used for the experiment: RLG7554 (rrnB P1, open circles), RLG7553 (rrnB P2, open squares) and RLG7555 (Pveg, black circles). A representative experiment is shown. The experiment was repeated three times with similar results.
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Figure 1: Changes in activity of selected B. subtilis promoters during outgrowth from stationary phase. Cells were grown in rich MOPS 20 amino acids medium until 3 h into stationary phase (time ‘0’). Subsequently, cells were diluted into fresh medium and RNA was extracted at the indicated time points. RNA transcribed from the tested promoter was quantitated and used as a measure of the promoter’s activity (‘Materials and Methods’ section). To simplify comparison of the promoters in terms of the proportional increase in their activity, the activities of the promoters were normalized to 1 at time 0. The actual relative activities of the promoters normalized to rrnB P2 (set as 1) at time 0 were as follows: rrnB P2 was 1, Pveg was 1.26 and rrnB P1 was 0.02. Thus, the activity of rrnB P1 was most repressed at time 0, allowing for the subsequent large increase in activity. Strains used for the experiment: RLG7554 (rrnB P1, open circles), RLG7553 (rrnB P2, open squares) and RLG7555 (Pveg, black circles). A representative experiment is shown. The experiment was repeated three times with similar results.

Mentions: Bacilllus subtilis contains 9–10 rRNA operons, depending on the strain (37). Escherichia coli contains seven rRNA operons (10). In both organisms, each operon is typically transcribed from a pair of promoters, P1 and P2. Both the P1 and P2 promoters in both organisms are sensitive to changes in the concentration of their iNTP. In E. coli, rrn P1 promoters exhibit more pronounced changes with growth rate and growth phase than rrn P2 promoters (10). In B. subtilis, similarly to E. coli, it was documented that an rrn P1 displayed more pronounced changes in activity with changes in growth rate than the corresponding rrn P2 (21). Here we show that this is also valid in B. subtilis for changes in growth phase, using rrnB P1 and rrnB P2 promoters and following their activity during outgrowth from stationary phase. We fused these promoters to a marker gene that gives rise to an in vivo unstable mRNA. We integrated the constructs in a single copy into the B. subtilis chromosome. The intracellular level of the mRNA was a measure of the promoter activity and was quantitated by RT–qPCR. During outgrowth, the rrnB P1 promoter increased its activity ∼100-fold, whereas rrnB P2 increased its activity ∼5-fold (Figure 1). A similar result was obtained with the B. subtilis rrnO P1 and P2 promoters (data not shown). Hence, for further studies, we decided to use an rrn P1 promoter because it displays more dramatic changes in activity than an rrn P2 promoter.Figure 1.


Rapid changes in gene expression: DNA determinants of promoter regulation by the concentration of the transcription initiating NTP in Bacillus subtilis.

Sojka L, Kouba T, Barvík I, Sanderová H, Maderová Z, Jonák J, Krásny L - Nucleic Acids Res. (2011)

Changes in activity of selected B. subtilis promoters during outgrowth from stationary phase. Cells were grown in rich MOPS 20 amino acids medium until 3 h into stationary phase (time ‘0’). Subsequently, cells were diluted into fresh medium and RNA was extracted at the indicated time points. RNA transcribed from the tested promoter was quantitated and used as a measure of the promoter’s activity (‘Materials and Methods’ section). To simplify comparison of the promoters in terms of the proportional increase in their activity, the activities of the promoters were normalized to 1 at time 0. The actual relative activities of the promoters normalized to rrnB P2 (set as 1) at time 0 were as follows: rrnB P2 was 1, Pveg was 1.26 and rrnB P1 was 0.02. Thus, the activity of rrnB P1 was most repressed at time 0, allowing for the subsequent large increase in activity. Strains used for the experiment: RLG7554 (rrnB P1, open circles), RLG7553 (rrnB P2, open squares) and RLG7555 (Pveg, black circles). A representative experiment is shown. The experiment was repeated three times with similar results.
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Related In: Results  -  Collection

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Figure 1: Changes in activity of selected B. subtilis promoters during outgrowth from stationary phase. Cells were grown in rich MOPS 20 amino acids medium until 3 h into stationary phase (time ‘0’). Subsequently, cells were diluted into fresh medium and RNA was extracted at the indicated time points. RNA transcribed from the tested promoter was quantitated and used as a measure of the promoter’s activity (‘Materials and Methods’ section). To simplify comparison of the promoters in terms of the proportional increase in their activity, the activities of the promoters were normalized to 1 at time 0. The actual relative activities of the promoters normalized to rrnB P2 (set as 1) at time 0 were as follows: rrnB P2 was 1, Pveg was 1.26 and rrnB P1 was 0.02. Thus, the activity of rrnB P1 was most repressed at time 0, allowing for the subsequent large increase in activity. Strains used for the experiment: RLG7554 (rrnB P1, open circles), RLG7553 (rrnB P2, open squares) and RLG7555 (Pveg, black circles). A representative experiment is shown. The experiment was repeated three times with similar results.
Mentions: Bacilllus subtilis contains 9–10 rRNA operons, depending on the strain (37). Escherichia coli contains seven rRNA operons (10). In both organisms, each operon is typically transcribed from a pair of promoters, P1 and P2. Both the P1 and P2 promoters in both organisms are sensitive to changes in the concentration of their iNTP. In E. coli, rrn P1 promoters exhibit more pronounced changes with growth rate and growth phase than rrn P2 promoters (10). In B. subtilis, similarly to E. coli, it was documented that an rrn P1 displayed more pronounced changes in activity with changes in growth rate than the corresponding rrn P2 (21). Here we show that this is also valid in B. subtilis for changes in growth phase, using rrnB P1 and rrnB P2 promoters and following their activity during outgrowth from stationary phase. We fused these promoters to a marker gene that gives rise to an in vivo unstable mRNA. We integrated the constructs in a single copy into the B. subtilis chromosome. The intracellular level of the mRNA was a measure of the promoter activity and was quantitated by RT–qPCR. During outgrowth, the rrnB P1 promoter increased its activity ∼100-fold, whereas rrnB P2 increased its activity ∼5-fold (Figure 1). A similar result was obtained with the B. subtilis rrnO P1 and P2 promoters (data not shown). Hence, for further studies, we decided to use an rrn P1 promoter because it displays more dramatic changes in activity than an rrn P2 promoter.Figure 1.

Bottom Line: An important small molecule effector is the initiating nucleoside triphosphate (iNTP).At some promoters, an increasing iNTP concentration stimulates promoter activity, while a decreasing concentration has the opposite effect.Overall, it seems that various sequence combinations can result in promoter regulation by [iNTP] in B. subtilis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics of Bacteria, Institute of Microbiology, Academy of Sciences of the Czech Republic.

ABSTRACT
In bacteria, rapid changes in gene expression can be achieved by affecting the activity of RNA polymerase with small molecule effectors during transcription initiation. An important small molecule effector is the initiating nucleoside triphosphate (iNTP). At some promoters, an increasing iNTP concentration stimulates promoter activity, while a decreasing concentration has the opposite effect. Ribosomal RNA (rRNA) promoters from Gram-positive Bacillus subtilis are regulated by the concentration of their iNTP. Yet, the sequences of these promoters do not emulate the sequence characteristics of [iNTP]-regulated rRNA promoters of Gram-negative Escherichia coli. Here, we identified the 3'-promoter region, corresponding to the transcription bubble, as key for B. subtilis rRNA promoter regulation via the concentration of the iNTP. Within this region, the conserved -5T (3 bp downstream from the -10 hexamer) is required for this regulation. Moreover, we identified a second class of [iNTP]-regulated promoters in B. subtilis where the sequence determinants are not limited to the transcription bubble region. Overall, it seems that various sequence combinations can result in promoter regulation by [iNTP] in B. subtilis. Finally, this study demonstrates how the same type of regulation can be achieved with strikingly different promoter sequences in phylogenetically distant species.

Show MeSH
Related in: MedlinePlus