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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.

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Comparison of the roles of GlrR and σ54 for expression of glmY from E. coli, S. typhimurium and Y. pseudotuberculosis. (A) EMSAs to test binding of E. coli GlrR protein to the glmY promoter regions of E. coli (−238 to +22), S. typhimurium (−242 to +22) and Y. pseudotuberculosis (−257 to +22). In addition to the glmY promoter fragments, 400 bp (panels 1 and 2) or 200 bp DNA fragments (panel 3) covering the lacZ promoter were present as internal controls. The sizes of the DNA size standard are given at the left. The apparent KD values are 360 nM for the E. coli glmY promoter, 230 nM for the Salmonella glmY promoter and 290 nM for the Y. pseudotuberculosis glmY promoter. (B) β-Galactosidase activities of E. coli strains carrying fusions of glmY’ from E. coli, S. typhimurium and Y. pseudotuberculosis to the lacZ reporter gene. In addition, these strains had the genotypes indicated in the legend. The following strains and transformants were tested (corresponding to the columns from left to right): Z197, Z206, Z206 + pBGG223, Z206 + pYG6, Z227, Z388, Z389, Z389 + pBGG223, Z389 + pYG6, Z446, Z362, Z363, Z363 + pBGG223, Z363 + pYG6 and Z444.
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Figure 2: Comparison of the roles of GlrR and σ54 for expression of glmY from E. coli, S. typhimurium and Y. pseudotuberculosis. (A) EMSAs to test binding of E. coli GlrR protein to the glmY promoter regions of E. coli (−238 to +22), S. typhimurium (−242 to +22) and Y. pseudotuberculosis (−257 to +22). In addition to the glmY promoter fragments, 400 bp (panels 1 and 2) or 200 bp DNA fragments (panel 3) covering the lacZ promoter were present as internal controls. The sizes of the DNA size standard are given at the left. The apparent KD values are 360 nM for the E. coli glmY promoter, 230 nM for the Salmonella glmY promoter and 290 nM for the Y. pseudotuberculosis glmY promoter. (B) β-Galactosidase activities of E. coli strains carrying fusions of glmY’ from E. coli, S. typhimurium and Y. pseudotuberculosis to the lacZ reporter gene. In addition, these strains had the genotypes indicated in the legend. The following strains and transformants were tested (corresponding to the columns from left to right): Z197, Z206, Z206 + pBGG223, Z206 + pYG6, Z227, Z388, Z389, Z389 + pBGG223, Z389 + pYG6, Z446, Z362, Z363, Z363 + pBGG223, Z363 + pYG6 and Z444.

Mentions: First, we wanted to verify if the putative σ54-dependent glmY promoters of S. typhimurium and Y. pseudotuberculosis are controlled by the response regulator GlrR. Therefore, we tested whether purified GlrR protein is able to bind to these promoters. EMSAs were carried out using purified GlrR protein from E. coli and DNA fragments covering the glmY promoter regions of these species. For comparison, binding of GlrR to the corresponding DNA fragment of E. coli was tested. Different concentrations of purified His-tagged GlrR protein were incubated with the various glmY promoter fragments, respectively. In order to verify binding specificity, an additional DNA fragment, which covered the lacZ promoter and had a size of either 400 or 200 bp was simultaneously present in these assays. Protein/DNA-complexes and unbound DNA were separated by polyacrylamide gel electrophoresis (Figure 2A). The glmY promoter fragments of all three species were shifted to distinct slower migrating bands indicating DNA/GlrR complexes, while the lacZ control fragments were not bound. Comparable protein concentrations were required to achieve binding, indicating that GlrR binds with similar affinities to all these glmY fragments. GlrR of E. coli shares 95% and 87% amino acid sequence identity with its homologs from S. typhimurium and Y. pseudotuberculosis, respectively. To confirm that the results obtained with the heterologous GlrR protein are valid, we additionally performed EMSAs using purified Y. pseudotuberculosis GlrR. This protein also bound the glmY promoter DNA fragments of both, Y. pseudotuberculosis and E. coli, with comparable affinities (Supplementary Figure S5). However, in comparison to GlrR from E. coli higher protein concentrations were required to achieve binding.Figure 2.


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)

Comparison of the roles of GlrR and σ54 for expression of glmY from E. coli, S. typhimurium and Y. pseudotuberculosis. (A) EMSAs to test binding of E. coli GlrR protein to the glmY promoter regions of E. coli (−238 to +22), S. typhimurium (−242 to +22) and Y. pseudotuberculosis (−257 to +22). In addition to the glmY promoter fragments, 400 bp (panels 1 and 2) or 200 bp DNA fragments (panel 3) covering the lacZ promoter were present as internal controls. The sizes of the DNA size standard are given at the left. The apparent KD values are 360 nM for the E. coli glmY promoter, 230 nM for the Salmonella glmY promoter and 290 nM for the Y. pseudotuberculosis glmY promoter. (B) β-Galactosidase activities of E. coli strains carrying fusions of glmY’ from E. coli, S. typhimurium and Y. pseudotuberculosis to the lacZ reporter gene. In addition, these strains had the genotypes indicated in the legend. The following strains and transformants were tested (corresponding to the columns from left to right): Z197, Z206, Z206 + pBGG223, Z206 + pYG6, Z227, Z388, Z389, Z389 + pBGG223, Z389 + pYG6, Z446, Z362, Z363, Z363 + pBGG223, Z363 + pYG6 and Z444.
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Figure 2: Comparison of the roles of GlrR and σ54 for expression of glmY from E. coli, S. typhimurium and Y. pseudotuberculosis. (A) EMSAs to test binding of E. coli GlrR protein to the glmY promoter regions of E. coli (−238 to +22), S. typhimurium (−242 to +22) and Y. pseudotuberculosis (−257 to +22). In addition to the glmY promoter fragments, 400 bp (panels 1 and 2) or 200 bp DNA fragments (panel 3) covering the lacZ promoter were present as internal controls. The sizes of the DNA size standard are given at the left. The apparent KD values are 360 nM for the E. coli glmY promoter, 230 nM for the Salmonella glmY promoter and 290 nM for the Y. pseudotuberculosis glmY promoter. (B) β-Galactosidase activities of E. coli strains carrying fusions of glmY’ from E. coli, S. typhimurium and Y. pseudotuberculosis to the lacZ reporter gene. In addition, these strains had the genotypes indicated in the legend. The following strains and transformants were tested (corresponding to the columns from left to right): Z197, Z206, Z206 + pBGG223, Z206 + pYG6, Z227, Z388, Z389, Z389 + pBGG223, Z389 + pYG6, Z446, Z362, Z363, Z363 + pBGG223, Z363 + pYG6 and Z444.
Mentions: First, we wanted to verify if the putative σ54-dependent glmY promoters of S. typhimurium and Y. pseudotuberculosis are controlled by the response regulator GlrR. Therefore, we tested whether purified GlrR protein is able to bind to these promoters. EMSAs were carried out using purified GlrR protein from E. coli and DNA fragments covering the glmY promoter regions of these species. For comparison, binding of GlrR to the corresponding DNA fragment of E. coli was tested. Different concentrations of purified His-tagged GlrR protein were incubated with the various glmY promoter fragments, respectively. In order to verify binding specificity, an additional DNA fragment, which covered the lacZ promoter and had a size of either 400 or 200 bp was simultaneously present in these assays. Protein/DNA-complexes and unbound DNA were separated by polyacrylamide gel electrophoresis (Figure 2A). The glmY promoter fragments of all three species were shifted to distinct slower migrating bands indicating DNA/GlrR complexes, while the lacZ control fragments were not bound. Comparable protein concentrations were required to achieve binding, indicating that GlrR binds with similar affinities to all these glmY fragments. GlrR of E. coli shares 95% and 87% amino acid sequence identity with its homologs from S. typhimurium and Y. pseudotuberculosis, respectively. To confirm that the results obtained with the heterologous GlrR protein are valid, we additionally performed EMSAs using purified Y. pseudotuberculosis GlrR. This protein also bound the glmY promoter DNA fragments of both, Y. pseudotuberculosis and E. coli, with comparable affinities (Supplementary Figure S5). However, in comparison to GlrR from E. coli higher protein concentrations were required to achieve binding.Figure 2.

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