Limits...
Common and divergent features in transcriptional control of the homologous small RNAs GlmY and GlmZ in Enterobacteriaceae.

Göpel Y, Lüttmann D, Heroven AK, Reichenbach B, Dersch P, Görke B - Nucleic Acids Res. (2010)

Bottom Line: However, in a subset of species such as E. coli this relationship is partially lost in favor of σ(70)-dependent transcription.In addition, we show that activity of the σ(54)-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter.Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August-University, Grisebachstrasse 8, 37077 Göttingen, Germany.

ABSTRACT
Small RNAs GlmY and GlmZ compose a cascade that feedback-regulates synthesis of enzyme GlmS in Enterobacteriaceae. Here, we analyzed the transcriptional regulation of glmY/glmZ from Yersinia pseudotuberculosis, Salmonella typhimurium and Escherichia coli, as representatives for other enterobacterial species, which exhibit similar promoter architectures. The GlmY and GlmZ sRNAs of Y. pseudotuberculosis are transcribed from σ(54)-promoters that require activation by the response regulator GlrR through binding to three conserved sites located upstream of the promoters. This also applies to glmY/glmZ of S. typhimurium and glmY of E. coli, but as a difference additional σ(70)-promoters overlap the σ(54)-promoters and initiate transcription at the same site. In contrast, E. coli glmZ is transcribed from a single σ(70)-promoter. Thus, transcription of glmY and glmZ is controlled by σ(54) and the two-component system GlrR/GlrK (QseF/QseE) in Y. pseudotuberculosis and presumably in many other Enterobacteria. However, in a subset of species such as E. coli this relationship is partially lost in favor of σ(70)-dependent transcription. In addition, we show that activity of the σ(54)-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter. Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.

Show MeSH

Related in: MedlinePlus

Phosphorylation increases activity of response regulator GlrR. (A) Effect of acetyl phosphate on the DNA binding activity of GlrR as revealed by EMSA. EMSAs were performed using purified E. coli GlrR and the E. coli glmY promoter fragment. To test the possible effect of phosphorylation on GlrR activity, the protein was pre-incubated at 37°C for 1 h in the absence (left panel) or presence (right panel) of 50 mM acetyl phosphate before continuing with the EMSA protocol. (B) A glutamate replacement of the phosphorylation site Asp56 in GlrR strongly up-regulates glmY expression. E. coli strain Z206 carrying a ΔglrR mutation and the E. coli glmY’-lacZ fusion on the chromosome was complemented with plasmids carrying E. coli wild-type glrR (pBGG389, column 2), glrR-D56A (pBGG398, column 3), glrR-D56E (pBGG399, column 4) or no gene (pBAD33, column 1) under PAra promoter control. Subsequently, the β-galactosidase activities were determined from these transformants.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3045617&req=5

Figure 7: Phosphorylation increases activity of response regulator GlrR. (A) Effect of acetyl phosphate on the DNA binding activity of GlrR as revealed by EMSA. EMSAs were performed using purified E. coli GlrR and the E. coli glmY promoter fragment. To test the possible effect of phosphorylation on GlrR activity, the protein was pre-incubated at 37°C for 1 h in the absence (left panel) or presence (right panel) of 50 mM acetyl phosphate before continuing with the EMSA protocol. (B) A glutamate replacement of the phosphorylation site Asp56 in GlrR strongly up-regulates glmY expression. E. coli strain Z206 carrying a ΔglrR mutation and the E. coli glmY’-lacZ fusion on the chromosome was complemented with plasmids carrying E. coli wild-type glrR (pBGG389, column 2), glrR-D56A (pBGG398, column 3), glrR-D56E (pBGG399, column 4) or no gene (pBAD33, column 1) under PAra promoter control. Subsequently, the β-galactosidase activities were determined from these transformants.

Mentions: GlrR contains a response regulatory domain including the conserved putative phosphorylation site aspartate 56 at its N-terminus. Phosphorylation of GlrR by its cognate kinase GlrK has been previously demonstrated in vitro (32). Furthermore, a ΔglrK mutation was shown to abolish activity of the σ54-promoter of glmY in E. coli, suggesting that GlrK controls activity of this promoter through modulation of the phosphorylation state of GlrR (22). In many TCS, the histidine kinase is capable of phosphorylating as well as dephosphorylating the response regulator. Phosphorylation of the response regulator results in structural changes, which in most cases activate the protein and stimulate interaction with the target DNA (33). In a few cases the dephosphorylated protein was shown to be active (34). We wanted to discriminate, whether phosphorylated or dephosphorylated GlrR is active. Therefore, we exploited the fact that many response regulators can autophosphorylate in vitro using small molecules such as acetyl phosphate as phosphoryl group donors (35). Therefore, EMSAs were carried out using the E. coli glmY promoter fragment and the E. coli GlrR protein that was pre-incubated with 50 mM acetyl phosphate for 1 h at 37°C prior to EMSA. Since ongoing incubation of GlrR at 37°C resulted in increasing inactivation of the protein (compare left panels in Figures 2A and 7A), a control experiment was performed in which GlrR was treated the same way but acetyl phosphate was omitted. These experiments revealed that binding affinity of GlrR was somewhat increased by the acetyl phosphate treatment relative to the control (compare panels in Figure 7A).Figure 7.


Common and divergent features in transcriptional control of the homologous small RNAs GlmY and GlmZ in Enterobacteriaceae.

Göpel Y, Lüttmann D, Heroven AK, Reichenbach B, Dersch P, Görke B - Nucleic Acids Res. (2010)

Phosphorylation increases activity of response regulator GlrR. (A) Effect of acetyl phosphate on the DNA binding activity of GlrR as revealed by EMSA. EMSAs were performed using purified E. coli GlrR and the E. coli glmY promoter fragment. To test the possible effect of phosphorylation on GlrR activity, the protein was pre-incubated at 37°C for 1 h in the absence (left panel) or presence (right panel) of 50 mM acetyl phosphate before continuing with the EMSA protocol. (B) A glutamate replacement of the phosphorylation site Asp56 in GlrR strongly up-regulates glmY expression. E. coli strain Z206 carrying a ΔglrR mutation and the E. coli glmY’-lacZ fusion on the chromosome was complemented with plasmids carrying E. coli wild-type glrR (pBGG389, column 2), glrR-D56A (pBGG398, column 3), glrR-D56E (pBGG399, column 4) or no gene (pBAD33, column 1) under PAra promoter control. Subsequently, the β-galactosidase activities were determined from these transformants.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045617&req=5

Figure 7: Phosphorylation increases activity of response regulator GlrR. (A) Effect of acetyl phosphate on the DNA binding activity of GlrR as revealed by EMSA. EMSAs were performed using purified E. coli GlrR and the E. coli glmY promoter fragment. To test the possible effect of phosphorylation on GlrR activity, the protein was pre-incubated at 37°C for 1 h in the absence (left panel) or presence (right panel) of 50 mM acetyl phosphate before continuing with the EMSA protocol. (B) A glutamate replacement of the phosphorylation site Asp56 in GlrR strongly up-regulates glmY expression. E. coli strain Z206 carrying a ΔglrR mutation and the E. coli glmY’-lacZ fusion on the chromosome was complemented with plasmids carrying E. coli wild-type glrR (pBGG389, column 2), glrR-D56A (pBGG398, column 3), glrR-D56E (pBGG399, column 4) or no gene (pBAD33, column 1) under PAra promoter control. Subsequently, the β-galactosidase activities were determined from these transformants.
Mentions: GlrR contains a response regulatory domain including the conserved putative phosphorylation site aspartate 56 at its N-terminus. Phosphorylation of GlrR by its cognate kinase GlrK has been previously demonstrated in vitro (32). Furthermore, a ΔglrK mutation was shown to abolish activity of the σ54-promoter of glmY in E. coli, suggesting that GlrK controls activity of this promoter through modulation of the phosphorylation state of GlrR (22). In many TCS, the histidine kinase is capable of phosphorylating as well as dephosphorylating the response regulator. Phosphorylation of the response regulator results in structural changes, which in most cases activate the protein and stimulate interaction with the target DNA (33). In a few cases the dephosphorylated protein was shown to be active (34). We wanted to discriminate, whether phosphorylated or dephosphorylated GlrR is active. Therefore, we exploited the fact that many response regulators can autophosphorylate in vitro using small molecules such as acetyl phosphate as phosphoryl group donors (35). Therefore, EMSAs were carried out using the E. coli glmY promoter fragment and the E. coli GlrR protein that was pre-incubated with 50 mM acetyl phosphate for 1 h at 37°C prior to EMSA. Since ongoing incubation of GlrR at 37°C resulted in increasing inactivation of the protein (compare left panels in Figures 2A and 7A), a control experiment was performed in which GlrR was treated the same way but acetyl phosphate was omitted. These experiments revealed that binding affinity of GlrR was somewhat increased by the acetyl phosphate treatment relative to the control (compare panels in Figure 7A).Figure 7.

Bottom Line: However, in a subset of species such as E. coli this relationship is partially lost in favor of σ(70)-dependent transcription.In addition, we show that activity of the σ(54)-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter.Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August-University, Grisebachstrasse 8, 37077 Göttingen, Germany.

ABSTRACT
Small RNAs GlmY and GlmZ compose a cascade that feedback-regulates synthesis of enzyme GlmS in Enterobacteriaceae. Here, we analyzed the transcriptional regulation of glmY/glmZ from Yersinia pseudotuberculosis, Salmonella typhimurium and Escherichia coli, as representatives for other enterobacterial species, which exhibit similar promoter architectures. The GlmY and GlmZ sRNAs of Y. pseudotuberculosis are transcribed from σ(54)-promoters that require activation by the response regulator GlrR through binding to three conserved sites located upstream of the promoters. This also applies to glmY/glmZ of S. typhimurium and glmY of E. coli, but as a difference additional σ(70)-promoters overlap the σ(54)-promoters and initiate transcription at the same site. In contrast, E. coli glmZ is transcribed from a single σ(70)-promoter. Thus, transcription of glmY and glmZ is controlled by σ(54) and the two-component system GlrR/GlrK (QseF/QseE) in Y. pseudotuberculosis and presumably in many other Enterobacteria. However, in a subset of species such as E. coli this relationship is partially lost in favor of σ(70)-dependent transcription. In addition, we show that activity of the σ(54)-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter. Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.

Show MeSH
Related in: MedlinePlus