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Initiating nucleotide identity determines efficiency of RNA synthesis from 6S RNA templates in Bacillus subtilis but not Escherichia coli.

Cabrera-Ostertag IJ, Cavanagh AT, Wassarman KM - Nucleic Acids Res. (2013)

Bottom Line: Specifically, initiation with guanosine triphosphate (GTP) is required for efficient pRNA synthesis, providing mechanistic insight into why 6S-2 RNA does not support robust pRNA synthesis as it initiates with adenosine triphosphate (ATP).Intriguingly, E. coli RNA polymerase does not have a strong preference for initiating nucleotide identity.These observations highlight an important difference in biochemical properties of B. subtilis and E. coli RNA polymerases, specifically in their ability to use RNA templates efficiently, which also may reflect the differences in GTP and ATP metabolism in these two organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA.

ABSTRACT
The 6S RNA is a non-coding small RNA that binds within the active site of housekeeping forms of RNA polymerases (e.g. Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis) and regulates transcription. Efficient release of RNA polymerase from 6S RNA regulation during outgrowth from stationary phase is dependent on use of 6S RNA as a template to generate a product RNA (pRNA). Interestingly, B. subtilis has two 6S RNAs, 6S-1 and 6S-2, but only 6S-1 RNA appears to be used efficiently as a template for pRNA synthesis during outgrowth. Here, we demonstrate that the identity of the initiating nucleotide is particularly important for the B. subtilis RNA polymerase to use RNA templates. Specifically, initiation with guanosine triphosphate (GTP) is required for efficient pRNA synthesis, providing mechanistic insight into why 6S-2 RNA does not support robust pRNA synthesis as it initiates with adenosine triphosphate (ATP). Intriguingly, E. coli RNA polymerase does not have a strong preference for initiating nucleotide identity. These observations highlight an important difference in biochemical properties of B. subtilis and E. coli RNA polymerases, specifically in their ability to use RNA templates efficiently, which also may reflect the differences in GTP and ATP metabolism in these two organisms.

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Efficient pRNA synthesis by BsEσA requires initiation with GTP. (A) Schematic of Ec6S RNA in a secondary structure supported by phylogenetic and experimental analyses (3,4). The +1 position is indicated by red N in the sequence, with changes and names of RNA mutants in the red box. For Ec6S(iUCC), the changes in position +1 and +2 are shown in the blue box. (B) pRNAs generated in vitro by BsEσA from various RNAs or no RNA (indicated at top) when containing 32P γ-GTP, 32P γ-ATP or 32P α-CTP (indicated by A, G or C label at top) were visualized on a denaturing gel. Lane M contains a 5′ end labeled oligonucleotide 19 nt in length for size comparison. Hash mark indicates a smear present in the 32P γ-ATP preparation, as it is present in the absence of RNA in lane 16. (C) Growth of B. subtilis cells as monitored by optical density at 595 nm (OD595) in an absorbance plate reader after stationary phase cells were diluted ∼1:500 into 2× YT medium. Growth was of B. subtilis ΔbsrAΔbsrB cells (KW590) containing plasmids pSP*-Ec6S(iATP) (dotted red), pSP*-Ec6S(iGTP) (blue), pSP*Ec6S(iCTP) (green), pSP*-Ec6S(iUTP) (brown) or pSP*-Ec6S(iUcc) (dotted pink). Data shown are from one representative experiment with three biological replicates. Similar results were observed in at least three experiments. Error bars correspond to ± standard deviations from the averages.
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gkt517-F2: Efficient pRNA synthesis by BsEσA requires initiation with GTP. (A) Schematic of Ec6S RNA in a secondary structure supported by phylogenetic and experimental analyses (3,4). The +1 position is indicated by red N in the sequence, with changes and names of RNA mutants in the red box. For Ec6S(iUCC), the changes in position +1 and +2 are shown in the blue box. (B) pRNAs generated in vitro by BsEσA from various RNAs or no RNA (indicated at top) when containing 32P γ-GTP, 32P γ-ATP or 32P α-CTP (indicated by A, G or C label at top) were visualized on a denaturing gel. Lane M contains a 5′ end labeled oligonucleotide 19 nt in length for size comparison. Hash mark indicates a smear present in the 32P γ-ATP preparation, as it is present in the absence of RNA in lane 16. (C) Growth of B. subtilis cells as monitored by optical density at 595 nm (OD595) in an absorbance plate reader after stationary phase cells were diluted ∼1:500 into 2× YT medium. Growth was of B. subtilis ΔbsrAΔbsrB cells (KW590) containing plasmids pSP*-Ec6S(iATP) (dotted red), pSP*-Ec6S(iGTP) (blue), pSP*Ec6S(iCTP) (green), pSP*-Ec6S(iUTP) (brown) or pSP*-Ec6S(iUcc) (dotted pink). Data shown are from one representative experiment with three biological replicates. Similar results were observed in at least three experiments. Error bars correspond to ± standard deviations from the averages.

Mentions: pRNA generated from Bs6S-1 RNA (pRNABs6S-1) initiates with GTP, whereas pRNA generated from Bs6S-2 RNA (pRNABs6S-2) initiates with ATP (16), leading us to hypothesize that initiating nucleotide identity might be important for efficiency of pRNA synthesis. Therefore, we next made mutant RNAs with changes in the template +1 position to direct Bs6S-1 RNA to initiate with ATP [Bs6S-1(iATP) RNA] or Bs6S-2 RNA to initiate with GTP [Bs6S-2(iGTP) RNA]. Previously, a similar RNA to Bs6S-1(iATP) RNA [i.e. 6S-1(C40U)] was shown to direct reduced pRNA synthesis in vivo (16). However, under in vitro conditions examined here, the behavior of both the Bs6S-1(iATP) and Bs6S-2(iGTP) mutants was complex with substantial changes in the level of abortive relative to full-length products making analysis of the specific effect(s) of iNTP identity difficult. Therefore, we chose to move to a heterologous system and examine the behavior of Ec6S RNA and mutant Ec6S RNAs to investigate the potential role of individual nucleotide positions one at a time (see Figure 2A). pRNA generated from wild-type Ec6S RNA (pRNAEc6S) initiates with ATP by EcEσ70 [(5,6) see Figure 3]. However, we observed that Ec6S RNA does not serve as an efficient template for pRNA synthesis by BsEσA (Figure 2B, lane 3), although this RNA binds to BsEσA efficiently under conditions tested (Supplementary Figure S1). Examination of a mutant EcRNA that changed the template position to direct initiation with GTP [Ec6S(iGTP)] revealed that this RNA does support efficient pRNA synthesis by BsEσA (Figure 2B, lane 6).Figure 2.


Initiating nucleotide identity determines efficiency of RNA synthesis from 6S RNA templates in Bacillus subtilis but not Escherichia coli.

Cabrera-Ostertag IJ, Cavanagh AT, Wassarman KM - Nucleic Acids Res. (2013)

Efficient pRNA synthesis by BsEσA requires initiation with GTP. (A) Schematic of Ec6S RNA in a secondary structure supported by phylogenetic and experimental analyses (3,4). The +1 position is indicated by red N in the sequence, with changes and names of RNA mutants in the red box. For Ec6S(iUCC), the changes in position +1 and +2 are shown in the blue box. (B) pRNAs generated in vitro by BsEσA from various RNAs or no RNA (indicated at top) when containing 32P γ-GTP, 32P γ-ATP or 32P α-CTP (indicated by A, G or C label at top) were visualized on a denaturing gel. Lane M contains a 5′ end labeled oligonucleotide 19 nt in length for size comparison. Hash mark indicates a smear present in the 32P γ-ATP preparation, as it is present in the absence of RNA in lane 16. (C) Growth of B. subtilis cells as monitored by optical density at 595 nm (OD595) in an absorbance plate reader after stationary phase cells were diluted ∼1:500 into 2× YT medium. Growth was of B. subtilis ΔbsrAΔbsrB cells (KW590) containing plasmids pSP*-Ec6S(iATP) (dotted red), pSP*-Ec6S(iGTP) (blue), pSP*Ec6S(iCTP) (green), pSP*-Ec6S(iUTP) (brown) or pSP*-Ec6S(iUcc) (dotted pink). Data shown are from one representative experiment with three biological replicates. Similar results were observed in at least three experiments. Error bars correspond to ± standard deviations from the averages.
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Related In: Results  -  Collection

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gkt517-F2: Efficient pRNA synthesis by BsEσA requires initiation with GTP. (A) Schematic of Ec6S RNA in a secondary structure supported by phylogenetic and experimental analyses (3,4). The +1 position is indicated by red N in the sequence, with changes and names of RNA mutants in the red box. For Ec6S(iUCC), the changes in position +1 and +2 are shown in the blue box. (B) pRNAs generated in vitro by BsEσA from various RNAs or no RNA (indicated at top) when containing 32P γ-GTP, 32P γ-ATP or 32P α-CTP (indicated by A, G or C label at top) were visualized on a denaturing gel. Lane M contains a 5′ end labeled oligonucleotide 19 nt in length for size comparison. Hash mark indicates a smear present in the 32P γ-ATP preparation, as it is present in the absence of RNA in lane 16. (C) Growth of B. subtilis cells as monitored by optical density at 595 nm (OD595) in an absorbance plate reader after stationary phase cells were diluted ∼1:500 into 2× YT medium. Growth was of B. subtilis ΔbsrAΔbsrB cells (KW590) containing plasmids pSP*-Ec6S(iATP) (dotted red), pSP*-Ec6S(iGTP) (blue), pSP*Ec6S(iCTP) (green), pSP*-Ec6S(iUTP) (brown) or pSP*-Ec6S(iUcc) (dotted pink). Data shown are from one representative experiment with three biological replicates. Similar results were observed in at least three experiments. Error bars correspond to ± standard deviations from the averages.
Mentions: pRNA generated from Bs6S-1 RNA (pRNABs6S-1) initiates with GTP, whereas pRNA generated from Bs6S-2 RNA (pRNABs6S-2) initiates with ATP (16), leading us to hypothesize that initiating nucleotide identity might be important for efficiency of pRNA synthesis. Therefore, we next made mutant RNAs with changes in the template +1 position to direct Bs6S-1 RNA to initiate with ATP [Bs6S-1(iATP) RNA] or Bs6S-2 RNA to initiate with GTP [Bs6S-2(iGTP) RNA]. Previously, a similar RNA to Bs6S-1(iATP) RNA [i.e. 6S-1(C40U)] was shown to direct reduced pRNA synthesis in vivo (16). However, under in vitro conditions examined here, the behavior of both the Bs6S-1(iATP) and Bs6S-2(iGTP) mutants was complex with substantial changes in the level of abortive relative to full-length products making analysis of the specific effect(s) of iNTP identity difficult. Therefore, we chose to move to a heterologous system and examine the behavior of Ec6S RNA and mutant Ec6S RNAs to investigate the potential role of individual nucleotide positions one at a time (see Figure 2A). pRNA generated from wild-type Ec6S RNA (pRNAEc6S) initiates with ATP by EcEσ70 [(5,6) see Figure 3]. However, we observed that Ec6S RNA does not serve as an efficient template for pRNA synthesis by BsEσA (Figure 2B, lane 3), although this RNA binds to BsEσA efficiently under conditions tested (Supplementary Figure S1). Examination of a mutant EcRNA that changed the template position to direct initiation with GTP [Ec6S(iGTP)] revealed that this RNA does support efficient pRNA synthesis by BsEσA (Figure 2B, lane 6).Figure 2.

Bottom Line: Specifically, initiation with guanosine triphosphate (GTP) is required for efficient pRNA synthesis, providing mechanistic insight into why 6S-2 RNA does not support robust pRNA synthesis as it initiates with adenosine triphosphate (ATP).Intriguingly, E. coli RNA polymerase does not have a strong preference for initiating nucleotide identity.These observations highlight an important difference in biochemical properties of B. subtilis and E. coli RNA polymerases, specifically in their ability to use RNA templates efficiently, which also may reflect the differences in GTP and ATP metabolism in these two organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA.

ABSTRACT
The 6S RNA is a non-coding small RNA that binds within the active site of housekeeping forms of RNA polymerases (e.g. Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis) and regulates transcription. Efficient release of RNA polymerase from 6S RNA regulation during outgrowth from stationary phase is dependent on use of 6S RNA as a template to generate a product RNA (pRNA). Interestingly, B. subtilis has two 6S RNAs, 6S-1 and 6S-2, but only 6S-1 RNA appears to be used efficiently as a template for pRNA synthesis during outgrowth. Here, we demonstrate that the identity of the initiating nucleotide is particularly important for the B. subtilis RNA polymerase to use RNA templates. Specifically, initiation with guanosine triphosphate (GTP) is required for efficient pRNA synthesis, providing mechanistic insight into why 6S-2 RNA does not support robust pRNA synthesis as it initiates with adenosine triphosphate (ATP). Intriguingly, E. coli RNA polymerase does not have a strong preference for initiating nucleotide identity. These observations highlight an important difference in biochemical properties of B. subtilis and E. coli RNA polymerases, specifically in their ability to use RNA templates efficiently, which also may reflect the differences in GTP and ATP metabolism in these two organisms.

Show MeSH
Related in: MedlinePlus