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Regulation of 6S RNA by pRNA synthesis is required for efficient recovery from stationary phase in E. coli and B. subtilis.

Cavanagh AT, Sperger JM, Wassarman KM - Nucleic Acids Res. (2011)

Bottom Line: Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA.The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally.Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.

View Article: PubMed Central - PubMed

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

ABSTRACT
6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ(70) during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ(70) to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.

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Characterization of Bs6S-1 and Bs6S-2 RNAs in vitro and in vivo. (A) pRNA generated in vitro from Bs6S-1:EσA (lane 2) or Bs6S-2:EσA (lane 3) complexes or with EσA alone (lane 4) was visualized on a denaturing gel. Reactions contained 0.05 mM NTPs including α-32P-CTP. Lane 1 is a 5′-end labeled oligonucleotide 19 nt in length for size comparison. (B) Bs6S-1 RNA (lanes 1–3) and Bs6S-2 RNA (lanes 4–6) association with EσA was monitored by migration in native gels. In vitro reactions contained RNA alone (lanes 1, 4); RNA and EσA (lanes 2, 5) or RNA, EσA and NTPs (0.05 mM) (lanes 3, 6). The locations of free RNA, RNA:pRNA duplexes and RNA:EσA migration are indicated. Note that Bs6S-1 RNA and Bs6S-2 RNA experiments were done in parallel, but run on separate gels as indicated by line between lanes 3 and 4. (C) Northern analysis of small RNA isolated from B. subtilis 168 cells (KW586) to examine in vivo-generated pRNAs in stationary phase (S; lane 3); or 2 (lane 4), 10 (lane 5) or 20 (lane 6) min after dilution of stationary phase cells into 2× YT medium. R1 (lane 2) and R2 (lane 1) contain synthetic RNAs corresponding to predicted pRNA6S-1 and pRNA6S-2, respectively, to test probing efficiency of LNA probes specific for pRNA6S-1 (top panel) or pRNA6S-2 (bottom panel).
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gkr1003-F3: Characterization of Bs6S-1 and Bs6S-2 RNAs in vitro and in vivo. (A) pRNA generated in vitro from Bs6S-1:EσA (lane 2) or Bs6S-2:EσA (lane 3) complexes or with EσA alone (lane 4) was visualized on a denaturing gel. Reactions contained 0.05 mM NTPs including α-32P-CTP. Lane 1 is a 5′-end labeled oligonucleotide 19 nt in length for size comparison. (B) Bs6S-1 RNA (lanes 1–3) and Bs6S-2 RNA (lanes 4–6) association with EσA was monitored by migration in native gels. In vitro reactions contained RNA alone (lanes 1, 4); RNA and EσA (lanes 2, 5) or RNA, EσA and NTPs (0.05 mM) (lanes 3, 6). The locations of free RNA, RNA:pRNA duplexes and RNA:EσA migration are indicated. Note that Bs6S-1 RNA and Bs6S-2 RNA experiments were done in parallel, but run on separate gels as indicated by line between lanes 3 and 4. (C) Northern analysis of small RNA isolated from B. subtilis 168 cells (KW586) to examine in vivo-generated pRNAs in stationary phase (S; lane 3); or 2 (lane 4), 10 (lane 5) or 20 (lane 6) min after dilution of stationary phase cells into 2× YT medium. R1 (lane 2) and R2 (lane 1) contain synthetic RNAs corresponding to predicted pRNA6S-1 and pRNA6S-2, respectively, to test probing efficiency of LNA probes specific for pRNA6S-1 (top panel) or pRNA6S-2 (bottom panel).

Mentions: Both Bs6S-1 and Bs6S-2 RNAs bind to EσA efficiently in vivo and in vitro (5), which raised the question of what distinguishes 6S-1 and 6S-2 RNA functions. Analysis of RNAs in wild-type B. subtilis by deep sequencing failed to observe a 6S-2 RNA-directed pRNA (pRNA6S-2), although 6S-1 RNA-directed pRNA (pRNA6S-1) was readily detected (34), suggesting that 6S-2 RNA may not be used as a template for pRNA synthesis. Alternatively, it is possible that pRNA6S-2 is unusually unstable relative to pRNA6S-1, or that growth conditions examined were not optimal for observing synthesis of pRNA6S-2. To fully assess the potential for 6S-2 RNA to direct pRNA synthesis, we turned to in vitro assays using purified B. subtilis EσA and RNA to allow easy manipulation of RNA levels and eliminate potential effects from differential stability of generated pRNAs. Incubation of Bs6S-1 RNA:EσA complexes with nucleotides resulted in production of readily detected pRNA6S-1 products (Figure 3A), indicating Bs6S-1 RNA can direct pRNA synthesis in agreement with similar work by others and deep sequencing analysis (22,34). In contrast, incubation of Bs6S-2 RNA:EσA complexes with nucleotides produced only minimal pRNA (Figure 3A), suggesting it is a poor template for pRNA synthesis.Figure 3.


Regulation of 6S RNA by pRNA synthesis is required for efficient recovery from stationary phase in E. coli and B. subtilis.

Cavanagh AT, Sperger JM, Wassarman KM - Nucleic Acids Res. (2011)

Characterization of Bs6S-1 and Bs6S-2 RNAs in vitro and in vivo. (A) pRNA generated in vitro from Bs6S-1:EσA (lane 2) or Bs6S-2:EσA (lane 3) complexes or with EσA alone (lane 4) was visualized on a denaturing gel. Reactions contained 0.05 mM NTPs including α-32P-CTP. Lane 1 is a 5′-end labeled oligonucleotide 19 nt in length for size comparison. (B) Bs6S-1 RNA (lanes 1–3) and Bs6S-2 RNA (lanes 4–6) association with EσA was monitored by migration in native gels. In vitro reactions contained RNA alone (lanes 1, 4); RNA and EσA (lanes 2, 5) or RNA, EσA and NTPs (0.05 mM) (lanes 3, 6). The locations of free RNA, RNA:pRNA duplexes and RNA:EσA migration are indicated. Note that Bs6S-1 RNA and Bs6S-2 RNA experiments were done in parallel, but run on separate gels as indicated by line between lanes 3 and 4. (C) Northern analysis of small RNA isolated from B. subtilis 168 cells (KW586) to examine in vivo-generated pRNAs in stationary phase (S; lane 3); or 2 (lane 4), 10 (lane 5) or 20 (lane 6) min after dilution of stationary phase cells into 2× YT medium. R1 (lane 2) and R2 (lane 1) contain synthetic RNAs corresponding to predicted pRNA6S-1 and pRNA6S-2, respectively, to test probing efficiency of LNA probes specific for pRNA6S-1 (top panel) or pRNA6S-2 (bottom panel).
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Related In: Results  -  Collection

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gkr1003-F3: Characterization of Bs6S-1 and Bs6S-2 RNAs in vitro and in vivo. (A) pRNA generated in vitro from Bs6S-1:EσA (lane 2) or Bs6S-2:EσA (lane 3) complexes or with EσA alone (lane 4) was visualized on a denaturing gel. Reactions contained 0.05 mM NTPs including α-32P-CTP. Lane 1 is a 5′-end labeled oligonucleotide 19 nt in length for size comparison. (B) Bs6S-1 RNA (lanes 1–3) and Bs6S-2 RNA (lanes 4–6) association with EσA was monitored by migration in native gels. In vitro reactions contained RNA alone (lanes 1, 4); RNA and EσA (lanes 2, 5) or RNA, EσA and NTPs (0.05 mM) (lanes 3, 6). The locations of free RNA, RNA:pRNA duplexes and RNA:EσA migration are indicated. Note that Bs6S-1 RNA and Bs6S-2 RNA experiments were done in parallel, but run on separate gels as indicated by line between lanes 3 and 4. (C) Northern analysis of small RNA isolated from B. subtilis 168 cells (KW586) to examine in vivo-generated pRNAs in stationary phase (S; lane 3); or 2 (lane 4), 10 (lane 5) or 20 (lane 6) min after dilution of stationary phase cells into 2× YT medium. R1 (lane 2) and R2 (lane 1) contain synthetic RNAs corresponding to predicted pRNA6S-1 and pRNA6S-2, respectively, to test probing efficiency of LNA probes specific for pRNA6S-1 (top panel) or pRNA6S-2 (bottom panel).
Mentions: Both Bs6S-1 and Bs6S-2 RNAs bind to EσA efficiently in vivo and in vitro (5), which raised the question of what distinguishes 6S-1 and 6S-2 RNA functions. Analysis of RNAs in wild-type B. subtilis by deep sequencing failed to observe a 6S-2 RNA-directed pRNA (pRNA6S-2), although 6S-1 RNA-directed pRNA (pRNA6S-1) was readily detected (34), suggesting that 6S-2 RNA may not be used as a template for pRNA synthesis. Alternatively, it is possible that pRNA6S-2 is unusually unstable relative to pRNA6S-1, or that growth conditions examined were not optimal for observing synthesis of pRNA6S-2. To fully assess the potential for 6S-2 RNA to direct pRNA synthesis, we turned to in vitro assays using purified B. subtilis EσA and RNA to allow easy manipulation of RNA levels and eliminate potential effects from differential stability of generated pRNAs. Incubation of Bs6S-1 RNA:EσA complexes with nucleotides resulted in production of readily detected pRNA6S-1 products (Figure 3A), indicating Bs6S-1 RNA can direct pRNA synthesis in agreement with similar work by others and deep sequencing analysis (22,34). In contrast, incubation of Bs6S-2 RNA:EσA complexes with nucleotides produced only minimal pRNA (Figure 3A), suggesting it is a poor template for pRNA synthesis.Figure 3.

Bottom Line: Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA.The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally.Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.

View Article: PubMed Central - PubMed

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

ABSTRACT
6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ(70) during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ(70) to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.

Show MeSH
Related in: MedlinePlus