Limits...
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.

Show MeSH

Related in: MedlinePlus

DNA elements of B. subtilis rrnB P1 required for its sensitivity to [iNTP] in vitro. (A) Sequence comparison of Pveg-rrnB P1 chimeric constructs. The sequence is highlighted with two shades of gray, indicating from where this sequence fragment comes: light gray, Pveg; dark gray, rrnB P1. (B) Multiple-round transcriptions as a function of GTP concentration: representative primary data and their graphical comparison for rrnB P1, Pveg and two chimeric constructs (Nos 4 and 9). The graph shows the 0–1000 µM interval. (C) Graphical comparison of KGTP values for construct Nos 1–9. KGTP values are shown above the bars. The values are the averages of three independent experiments. The error bars in this and all subsequent figures represent ±SD of the mean. For construct Nos 1, 4, 7 and 9, distinct bar fill patterns were used to facilitate orientation in this figure. (D) Open complex stability of construct Nos 1–9. Half-lives (t1/2) are indicated above the bars. (E) Sequence alignment of the region between −10 and +1 of B. subtilis rrn P1 promoters. The 100% conserved −5T is indicated in red. (F) Graphical representation of KGTP values for constructs testing the role of −5T. KGTP values are shown above the bars.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113569&req=5

Figure 3: DNA elements of B. subtilis rrnB P1 required for its sensitivity to [iNTP] in vitro. (A) Sequence comparison of Pveg-rrnB P1 chimeric constructs. The sequence is highlighted with two shades of gray, indicating from where this sequence fragment comes: light gray, Pveg; dark gray, rrnB P1. (B) Multiple-round transcriptions as a function of GTP concentration: representative primary data and their graphical comparison for rrnB P1, Pveg and two chimeric constructs (Nos 4 and 9). The graph shows the 0–1000 µM interval. (C) Graphical comparison of KGTP values for construct Nos 1–9. KGTP values are shown above the bars. The values are the averages of three independent experiments. The error bars in this and all subsequent figures represent ±SD of the mean. For construct Nos 1, 4, 7 and 9, distinct bar fill patterns were used to facilitate orientation in this figure. (D) Open complex stability of construct Nos 1–9. Half-lives (t1/2) are indicated above the bars. (E) Sequence alignment of the region between −10 and +1 of B. subtilis rrn P1 promoters. The 100% conserved −5T is indicated in red. (F) Graphical representation of KGTP values for constructs testing the role of −5T. KGTP values are shown above the bars.

Mentions: We wished to test whether RNAP from E. coli would indeed display [iNTP]-insensitive behavior at Bsu-rrnB P1, as was predicted based on its sequence. First, we verified that E. coli RNAP required a relatively high concentration of its iNTP (ATP) at Eco-rrnB P1 to reach maximal transcription, displaying typical [iNTP]-sensitive behavior in vitro (Figure 2B). On the contrary and as predicted, E. coli RNAP required a relatively low concentration of the iNTP at Bsu-rrnB P1 (Figure 2B). As a control, we showed that B. subtilis RNAP was sensitive to a wide concentration range of the iNTP at Bsu-rrnB P1 (Figure 2B). Finally, B. subtilis RNAP did not utilize Eco-rrnB P1 as a promoter (Figure 2B) and Pveg was recognized as an [iNTP]-insensitive promoter with RNAPs from both E. coli (data not shown) and B. subtilis (Figure 3B).Figure 3.


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)

DNA elements of B. subtilis rrnB P1 required for its sensitivity to [iNTP] in vitro. (A) Sequence comparison of Pveg-rrnB P1 chimeric constructs. The sequence is highlighted with two shades of gray, indicating from where this sequence fragment comes: light gray, Pveg; dark gray, rrnB P1. (B) Multiple-round transcriptions as a function of GTP concentration: representative primary data and their graphical comparison for rrnB P1, Pveg and two chimeric constructs (Nos 4 and 9). The graph shows the 0–1000 µM interval. (C) Graphical comparison of KGTP values for construct Nos 1–9. KGTP values are shown above the bars. The values are the averages of three independent experiments. The error bars in this and all subsequent figures represent ±SD of the mean. For construct Nos 1, 4, 7 and 9, distinct bar fill patterns were used to facilitate orientation in this figure. (D) Open complex stability of construct Nos 1–9. Half-lives (t1/2) are indicated above the bars. (E) Sequence alignment of the region between −10 and +1 of B. subtilis rrn P1 promoters. The 100% conserved −5T is indicated in red. (F) Graphical representation of KGTP values for constructs testing the role of −5T. KGTP values are shown above the bars.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113569&req=5

Figure 3: DNA elements of B. subtilis rrnB P1 required for its sensitivity to [iNTP] in vitro. (A) Sequence comparison of Pveg-rrnB P1 chimeric constructs. The sequence is highlighted with two shades of gray, indicating from where this sequence fragment comes: light gray, Pveg; dark gray, rrnB P1. (B) Multiple-round transcriptions as a function of GTP concentration: representative primary data and their graphical comparison for rrnB P1, Pveg and two chimeric constructs (Nos 4 and 9). The graph shows the 0–1000 µM interval. (C) Graphical comparison of KGTP values for construct Nos 1–9. KGTP values are shown above the bars. The values are the averages of three independent experiments. The error bars in this and all subsequent figures represent ±SD of the mean. For construct Nos 1, 4, 7 and 9, distinct bar fill patterns were used to facilitate orientation in this figure. (D) Open complex stability of construct Nos 1–9. Half-lives (t1/2) are indicated above the bars. (E) Sequence alignment of the region between −10 and +1 of B. subtilis rrn P1 promoters. The 100% conserved −5T is indicated in red. (F) Graphical representation of KGTP values for constructs testing the role of −5T. KGTP values are shown above the bars.
Mentions: We wished to test whether RNAP from E. coli would indeed display [iNTP]-insensitive behavior at Bsu-rrnB P1, as was predicted based on its sequence. First, we verified that E. coli RNAP required a relatively high concentration of its iNTP (ATP) at Eco-rrnB P1 to reach maximal transcription, displaying typical [iNTP]-sensitive behavior in vitro (Figure 2B). On the contrary and as predicted, E. coli RNAP required a relatively low concentration of the iNTP at Bsu-rrnB P1 (Figure 2B). As a control, we showed that B. subtilis RNAP was sensitive to a wide concentration range of the iNTP at Bsu-rrnB P1 (Figure 2B). Finally, B. subtilis RNAP did not utilize Eco-rrnB P1 as a promoter (Figure 2B) and Pveg was recognized as an [iNTP]-insensitive promoter with RNAPs from both E. coli (data not shown) and B. subtilis (Figure 3B).Figure 3.

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