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

6S(M68) RNA regulates transcription in E. coli similarly to 6S RNA. β-Galactosidase activity of promoter-lacZ reporter genes (rsdP2, lacUV5 or bolA) in E. coli ssrS1 cells expressing wild-type 6S RNA (gray bars); 6S(M6) RNA (black bars); 6S(M68) RNA (white bars); or 6S(M6+M68) RNA (hatched bars) from pKK-6S derived plasmids is shown. Data shown are averages of three independent experiments with three biological replicates per experiment. Error bars correspond to ± standard deviations from the averages.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1003-F5: 6S(M68) RNA regulates transcription in E. coli similarly to 6S RNA. β-Galactosidase activity of promoter-lacZ reporter genes (rsdP2, lacUV5 or bolA) in E. coli ssrS1 cells expressing wild-type 6S RNA (gray bars); 6S(M6) RNA (black bars); 6S(M68) RNA (white bars); or 6S(M6+M68) RNA (hatched bars) from pKK-6S derived plasmids is shown. Data shown are averages of three independent experiments with three biological replicates per experiment. Error bars correspond to ± standard deviations from the averages.

Mentions: Over the last decade, several key features important for 6S RNA and Eσ70 interactions have been identified. The 6S RNA is largely double-stranded with a central single-stranded region that is reminiscent of the conformation of DNA in the open complex during transcription initiation (Figure 1; 5,6). This structure directs 6S RNA binding to Eσ70 in the active site in a manner similar to the interactions between promoter DNA and Eσ70. 6S RNA blocks the ability of Eσ70 to bind to DNA, leading to downregulation of transcription at many σ70-dependent promoters, although other σ70-dependent promoters are insensitive to 6S RNA even during late stationary phase when the vast majority of Eσ70 is found in a complex with 6S RNA (7–11). σ70-dependent promoters are recognized through two sequence hexamers, the −10 element and the −35 element, which are recognized primarily by σ70 region 2.4 and 4.2, respectively (12). In contrast to how 6S RNA binds in the active site of Eσ70, the ‘upstream’ region of 6S RNA does not mimic a −35 element and many residues within region 4.2 of σ70 contribute differentially to 6S RNA or DNA binding (13). Interestingly, promoters with weak −35 elements are sensitive to 6S RNA regulation (10) suggesting region 4.2 of σ70 may be a primary site for competition between 6S RNA and promoter binding.Figure 1.


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)

6S(M68) RNA regulates transcription in E. coli similarly to 6S RNA. β-Galactosidase activity of promoter-lacZ reporter genes (rsdP2, lacUV5 or bolA) in E. coli ssrS1 cells expressing wild-type 6S RNA (gray bars); 6S(M6) RNA (black bars); 6S(M68) RNA (white bars); or 6S(M6+M68) RNA (hatched bars) from pKK-6S derived plasmids is shown. Data shown are averages of three independent experiments with three biological replicates per experiment. Error bars correspond to ± standard deviations from the averages.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1003-F5: 6S(M68) RNA regulates transcription in E. coli similarly to 6S RNA. β-Galactosidase activity of promoter-lacZ reporter genes (rsdP2, lacUV5 or bolA) in E. coli ssrS1 cells expressing wild-type 6S RNA (gray bars); 6S(M6) RNA (black bars); 6S(M68) RNA (white bars); or 6S(M6+M68) RNA (hatched bars) from pKK-6S derived plasmids is shown. Data shown are averages of three independent experiments with three biological replicates per experiment. Error bars correspond to ± standard deviations from the averages.
Mentions: Over the last decade, several key features important for 6S RNA and Eσ70 interactions have been identified. The 6S RNA is largely double-stranded with a central single-stranded region that is reminiscent of the conformation of DNA in the open complex during transcription initiation (Figure 1; 5,6). This structure directs 6S RNA binding to Eσ70 in the active site in a manner similar to the interactions between promoter DNA and Eσ70. 6S RNA blocks the ability of Eσ70 to bind to DNA, leading to downregulation of transcription at many σ70-dependent promoters, although other σ70-dependent promoters are insensitive to 6S RNA even during late stationary phase when the vast majority of Eσ70 is found in a complex with 6S RNA (7–11). σ70-dependent promoters are recognized through two sequence hexamers, the −10 element and the −35 element, which are recognized primarily by σ70 region 2.4 and 4.2, respectively (12). In contrast to how 6S RNA binds in the active site of Eσ70, the ‘upstream’ region of 6S RNA does not mimic a −35 element and many residues within region 4.2 of σ70 contribute differentially to 6S RNA or DNA binding (13). Interestingly, promoters with weak −35 elements are sensitive to 6S RNA regulation (10) suggesting region 4.2 of σ70 may be a primary site for competition between 6S RNA and promoter binding.Figure 1.

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