Limits...
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.

Show MeSH

Related in: MedlinePlus

Characterization of E. coli 6S(M68) RNA in vitro and in vivo. (A) RNA association with Eσ70 was monitored by native gel electrophoresis. 6S (lanes 1–3); 6S(M68) (lanes 4–6); and 6S(M5) (lanes 7–9) RNAs were examined. In vitro reactions contained RNA alone (lanes 1, 4 and 7); RNA and Eσ70 (lanes 2, 5 and 8) or RNA, Eσ70 and NTPs (lanes 3, 6 and 9). The locations of free RNA, 6S RNA:pRNA duplexes and RNA:Eσ70 migration are indicated. (B) pRNA generated by Eσ70in vitro from 6S RNA (lane 2); 6S(M68) RNA (lane 4); or 6S(M6+M68) RNA (lane 5) or Eσ70 alone (lane 3) was visualized on a denaturing gel. Lane 1 is a 5′-end labeled oligonucleotide 19 nt in length for size comparison. (C) Northern analysis of small RNA isolated from ssrS1 E. coli cells containing pKK-6S (lanes 3–5); pKK-6S(M68) (lanes 6–8), or an empty vector control (pKK-Cm, lanes 9–11) to examine in vivo generated pRNAs in stationary phase (S; 18 h) or after dilution of stationary phase cells into LB + Cm and continued incubation for 2 or 10 min as indicated. R1 (lane 2) and R2 (lane 1) contain synthetic RNAs corresponding to pRNAEc6S and the predicted pRNAEcM68, respectively, to test probing efficiency of the LNA probe.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3299989&req=5

gkr1003-F4: Characterization of E. coli 6S(M68) RNA in vitro and in vivo. (A) RNA association with Eσ70 was monitored by native gel electrophoresis. 6S (lanes 1–3); 6S(M68) (lanes 4–6); and 6S(M5) (lanes 7–9) RNAs were examined. In vitro reactions contained RNA alone (lanes 1, 4 and 7); RNA and Eσ70 (lanes 2, 5 and 8) or RNA, Eσ70 and NTPs (lanes 3, 6 and 9). The locations of free RNA, 6S RNA:pRNA duplexes and RNA:Eσ70 migration are indicated. (B) pRNA generated by Eσ70in vitro from 6S RNA (lane 2); 6S(M68) RNA (lane 4); or 6S(M6+M68) RNA (lane 5) or Eσ70 alone (lane 3) was visualized on a denaturing gel. Lane 1 is a 5′-end labeled oligonucleotide 19 nt in length for size comparison. (C) Northern analysis of small RNA isolated from ssrS1 E. coli cells containing pKK-6S (lanes 3–5); pKK-6S(M68) (lanes 6–8), or an empty vector control (pKK-Cm, lanes 9–11) to examine in vivo generated pRNAs in stationary phase (S; 18 h) or after dilution of stationary phase cells into LB + Cm and continued incubation for 2 or 10 min as indicated. R1 (lane 2) and R2 (lane 1) contain synthetic RNAs corresponding to pRNAEc6S and the predicted pRNAEcM68, respectively, to test probing efficiency of the LNA probe.

Mentions: 6S(M68) RNA was characterized in a series of in vitro and in vivo assays to test if it mimicked Bs6S-2 RNA behavior. In vitro binding with purified components demonstrated that 6S(M68) RNA bound to Eσ70 similar to wild-type Ec6S RNA in vitro, while the inactive 6S(M5) RNA did not (Figure 4A, compare lanes 2, 5 and 8). Incubation of wild-type Ec6S RNA:Eσ70 complexes with nucleotides under conditions that initiate pRNA synthesis has been shown to result in release of Ec6S RNA in a duplex with pRNA (8,21), which can be observed by native gel electrophoresis as a reduction in Ec6S RNA:Eσ70 complexes and the formation of Ec6S RNA:pRNA duplexes (Figure 4A, compare lanes 2 and 3). In contrast, incubation of the 6S(M68) RNA:Eσ70 complex with nucleotides did not lead to efficient release of 6S(M68) RNA:pRNA or reduction in 6S(M68) RNA:Eσ70 complexes (Figure 4A, lane 6). Direct analysis of in vitro-generated pRNA verified that 6S(M68) RNA is a poor template for pRNA synthesis (Figure 4B). A faint, smaller RNA was detected in vitro in the presence of 6S(M68) RNA that was not observed in the absence of RNA or with the inactive 6S(M6+M68) RNA, suggesting a low-level reaction can take place. However, any release of 6S(M68) RNA from Eσ70 is very low compared to wild-type 6S RNA (Figure 4A).Figure 4.


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 E. coli 6S(M68) RNA in vitro and in vivo. (A) RNA association with Eσ70 was monitored by native gel electrophoresis. 6S (lanes 1–3); 6S(M68) (lanes 4–6); and 6S(M5) (lanes 7–9) RNAs were examined. In vitro reactions contained RNA alone (lanes 1, 4 and 7); RNA and Eσ70 (lanes 2, 5 and 8) or RNA, Eσ70 and NTPs (lanes 3, 6 and 9). The locations of free RNA, 6S RNA:pRNA duplexes and RNA:Eσ70 migration are indicated. (B) pRNA generated by Eσ70in vitro from 6S RNA (lane 2); 6S(M68) RNA (lane 4); or 6S(M6+M68) RNA (lane 5) or Eσ70 alone (lane 3) was visualized on a denaturing gel. Lane 1 is a 5′-end labeled oligonucleotide 19 nt in length for size comparison. (C) Northern analysis of small RNA isolated from ssrS1 E. coli cells containing pKK-6S (lanes 3–5); pKK-6S(M68) (lanes 6–8), or an empty vector control (pKK-Cm, lanes 9–11) to examine in vivo generated pRNAs in stationary phase (S; 18 h) or after dilution of stationary phase cells into LB + Cm and continued incubation for 2 or 10 min as indicated. R1 (lane 2) and R2 (lane 1) contain synthetic RNAs corresponding to pRNAEc6S and the predicted pRNAEcM68, respectively, to test probing efficiency of the LNA probe.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3299989&req=5

gkr1003-F4: Characterization of E. coli 6S(M68) RNA in vitro and in vivo. (A) RNA association with Eσ70 was monitored by native gel electrophoresis. 6S (lanes 1–3); 6S(M68) (lanes 4–6); and 6S(M5) (lanes 7–9) RNAs were examined. In vitro reactions contained RNA alone (lanes 1, 4 and 7); RNA and Eσ70 (lanes 2, 5 and 8) or RNA, Eσ70 and NTPs (lanes 3, 6 and 9). The locations of free RNA, 6S RNA:pRNA duplexes and RNA:Eσ70 migration are indicated. (B) pRNA generated by Eσ70in vitro from 6S RNA (lane 2); 6S(M68) RNA (lane 4); or 6S(M6+M68) RNA (lane 5) or Eσ70 alone (lane 3) was visualized on a denaturing gel. Lane 1 is a 5′-end labeled oligonucleotide 19 nt in length for size comparison. (C) Northern analysis of small RNA isolated from ssrS1 E. coli cells containing pKK-6S (lanes 3–5); pKK-6S(M68) (lanes 6–8), or an empty vector control (pKK-Cm, lanes 9–11) to examine in vivo generated pRNAs in stationary phase (S; 18 h) or after dilution of stationary phase cells into LB + Cm and continued incubation for 2 or 10 min as indicated. R1 (lane 2) and R2 (lane 1) contain synthetic RNAs corresponding to pRNAEc6S and the predicted pRNAEcM68, respectively, to test probing efficiency of the LNA probe.
Mentions: 6S(M68) RNA was characterized in a series of in vitro and in vivo assays to test if it mimicked Bs6S-2 RNA behavior. In vitro binding with purified components demonstrated that 6S(M68) RNA bound to Eσ70 similar to wild-type Ec6S RNA in vitro, while the inactive 6S(M5) RNA did not (Figure 4A, compare lanes 2, 5 and 8). Incubation of wild-type Ec6S RNA:Eσ70 complexes with nucleotides under conditions that initiate pRNA synthesis has been shown to result in release of Ec6S RNA in a duplex with pRNA (8,21), which can be observed by native gel electrophoresis as a reduction in Ec6S RNA:Eσ70 complexes and the formation of Ec6S RNA:pRNA duplexes (Figure 4A, compare lanes 2 and 3). In contrast, incubation of the 6S(M68) RNA:Eσ70 complex with nucleotides did not lead to efficient release of 6S(M68) RNA:pRNA or reduction in 6S(M68) RNA:Eσ70 complexes (Figure 4A, lane 6). Direct analysis of in vitro-generated pRNA verified that 6S(M68) RNA is a poor template for pRNA synthesis (Figure 4B). A faint, smaller RNA was detected in vitro in the presence of 6S(M68) RNA that was not observed in the absence of RNA or with the inactive 6S(M6+M68) RNA, suggesting a low-level reaction can take place. However, any release of 6S(M68) RNA from Eσ70 is very low compared to wild-type 6S RNA (Figure 4A).Figure 4.

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