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The mechanism for RNA recognition by ANTAR regulators of gene expression.

Ramesh A, DebRoy S, Goodson JR, Fox KA, Faz H, Garsin DA, Winkler WC - PLoS Genet. (2012)

Bottom Line: The novel antiterminator structure consists of two small hairpins with highly conserved terminal loop residues, both features being essential for successful antitermination.Despite the unrelatedness of the species in which they are found, the majority of the ANTAR-associated genes are thematically related to nitrogen management.These data suggest that the central tenets for gene regulation by ANTAR antitermination occur widely in nature to specifically control nitrogen metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

ABSTRACT
ANTAR proteins are widespread bacterial regulatory proteins that have RNA-binding output domains and utilize antitermination to control gene expression at the post-initiation level. An ANTAR protein, EutV, regulates the ethanolamine-utilization genes (eut) in Enterococcus faecalis. Using this system, we present genetic and biochemical evidence of a general mechanism of antitermination used by ANTARs, including details of the antiterminator structure. The novel antiterminator structure consists of two small hairpins with highly conserved terminal loop residues, both features being essential for successful antitermination. The ANTAR protein dimerizes and associates with its substrate RNA in response to signal-induced phosphorylation. Furthermore, bioinformatic searches using this conserved antiterminator motif identified many new ANTAR target RNAs in phylogenetically diverse bacterial species, some comprising complex regulons. Despite the unrelatedness of the species in which they are found, the majority of the ANTAR-associated genes are thematically related to nitrogen management. These data suggest that the central tenets for gene regulation by ANTAR antitermination occur widely in nature to specifically control nitrogen metabolism.

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Multiple input signals, along with protein and RNA elements, regulate the eut locus in Enterococcus faecalis.A) The organization of the E. faecalis eut locus is shown schematically. The EutV/EutW two component regulatory system responds to ethanolamine to stimulate EutW autophosphorylation followed by phosphoryl transfer to EutV [15], [17]. Phosphorylated EutV is hypothesized to prevent formation of four different intrinsic terminator sites (red) within the eut pathway [17], [18]. Additionally, an AdoCbl-sensing riboswitch is located upstream of eutG[14], [16]. B) Expression of lacZ translational fusions to the eutP 5′ leader region is shown as bar graphs and is described in the text. Each fusion is represented by a color with the darker shade indicating the wild type background and the lighter shade designating the eutVW background. Presence of both AdoCbl and ethanolamine was required for induction of eutP containing the wild-type leader sequence (blue). Deletion of the EutV/EutW two-component regulatory system abolished eutP induction (light blue). Deletion of the terminator in the leader, eutPΔT abolished EutV/EutW dependency (red/light red). The vector control in both backgrounds is shown in black/gray. (C) Expression of lacZ translational fusions to the eutS 5′ leader region. Color scheme is described in the figure.
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pgen-1002666-g002: Multiple input signals, along with protein and RNA elements, regulate the eut locus in Enterococcus faecalis.A) The organization of the E. faecalis eut locus is shown schematically. The EutV/EutW two component regulatory system responds to ethanolamine to stimulate EutW autophosphorylation followed by phosphoryl transfer to EutV [15], [17]. Phosphorylated EutV is hypothesized to prevent formation of four different intrinsic terminator sites (red) within the eut pathway [17], [18]. Additionally, an AdoCbl-sensing riboswitch is located upstream of eutG[14], [16]. B) Expression of lacZ translational fusions to the eutP 5′ leader region is shown as bar graphs and is described in the text. Each fusion is represented by a color with the darker shade indicating the wild type background and the lighter shade designating the eutVW background. Presence of both AdoCbl and ethanolamine was required for induction of eutP containing the wild-type leader sequence (blue). Deletion of the EutV/EutW two-component regulatory system abolished eutP induction (light blue). Deletion of the terminator in the leader, eutPΔT abolished EutV/EutW dependency (red/light red). The vector control in both backgrounds is shown in black/gray. (C) Expression of lacZ translational fusions to the eutS 5′ leader region. Color scheme is described in the figure.

Mentions: EutV, a representative of ANTAR-containing response regulators, was discovered to regulate the ethanolamine utilization operon (eut) in Enterococcus faecalis and this mode of regulation appears to be conserved in many Firmicutes that contain eut operons [15]–[17]. For E. faecalis, the corresponding sensor kinase, EutW, undergoes autophosphorylation in response to ethanolamine whereupon the phosphoryl group is transferred to EutV [15], [17]. Phosphorylated EutV is postulated to disrupt terminator sites located just upstream of each of the genes eutP, eutG, eutS and eutA (Figure 2A); its association is therefore predicted to activate downstream gene expression [15]–[17]. These locations within the eut operon were found to share a common 13-nucleotide sequence (AGCAANGRRGCUY) overlapping the 5′-proximal portion of their corresponding intrinsic terminator elements. We previously proposed that these sites could serve as part of the recognition sequence for ANTAR-based regulators in order to promote antitermination and allow production of the downstream transcript [17]. Recent work investigating the regulation of eutG in E. faecalis supports the model that antitermination occurs at this consensus sequence [18]. However, no functional studies have yet identified the sequence or structural features that are specifically important for antitermination in the eut system or any other system that utilizes ANTAR-based regulatory proteins.


The mechanism for RNA recognition by ANTAR regulators of gene expression.

Ramesh A, DebRoy S, Goodson JR, Fox KA, Faz H, Garsin DA, Winkler WC - PLoS Genet. (2012)

Multiple input signals, along with protein and RNA elements, regulate the eut locus in Enterococcus faecalis.A) The organization of the E. faecalis eut locus is shown schematically. The EutV/EutW two component regulatory system responds to ethanolamine to stimulate EutW autophosphorylation followed by phosphoryl transfer to EutV [15], [17]. Phosphorylated EutV is hypothesized to prevent formation of four different intrinsic terminator sites (red) within the eut pathway [17], [18]. Additionally, an AdoCbl-sensing riboswitch is located upstream of eutG[14], [16]. B) Expression of lacZ translational fusions to the eutP 5′ leader region is shown as bar graphs and is described in the text. Each fusion is represented by a color with the darker shade indicating the wild type background and the lighter shade designating the eutVW background. Presence of both AdoCbl and ethanolamine was required for induction of eutP containing the wild-type leader sequence (blue). Deletion of the EutV/EutW two-component regulatory system abolished eutP induction (light blue). Deletion of the terminator in the leader, eutPΔT abolished EutV/EutW dependency (red/light red). The vector control in both backgrounds is shown in black/gray. (C) Expression of lacZ translational fusions to the eutS 5′ leader region. Color scheme is described in the figure.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369931&req=5

pgen-1002666-g002: Multiple input signals, along with protein and RNA elements, regulate the eut locus in Enterococcus faecalis.A) The organization of the E. faecalis eut locus is shown schematically. The EutV/EutW two component regulatory system responds to ethanolamine to stimulate EutW autophosphorylation followed by phosphoryl transfer to EutV [15], [17]. Phosphorylated EutV is hypothesized to prevent formation of four different intrinsic terminator sites (red) within the eut pathway [17], [18]. Additionally, an AdoCbl-sensing riboswitch is located upstream of eutG[14], [16]. B) Expression of lacZ translational fusions to the eutP 5′ leader region is shown as bar graphs and is described in the text. Each fusion is represented by a color with the darker shade indicating the wild type background and the lighter shade designating the eutVW background. Presence of both AdoCbl and ethanolamine was required for induction of eutP containing the wild-type leader sequence (blue). Deletion of the EutV/EutW two-component regulatory system abolished eutP induction (light blue). Deletion of the terminator in the leader, eutPΔT abolished EutV/EutW dependency (red/light red). The vector control in both backgrounds is shown in black/gray. (C) Expression of lacZ translational fusions to the eutS 5′ leader region. Color scheme is described in the figure.
Mentions: EutV, a representative of ANTAR-containing response regulators, was discovered to regulate the ethanolamine utilization operon (eut) in Enterococcus faecalis and this mode of regulation appears to be conserved in many Firmicutes that contain eut operons [15]–[17]. For E. faecalis, the corresponding sensor kinase, EutW, undergoes autophosphorylation in response to ethanolamine whereupon the phosphoryl group is transferred to EutV [15], [17]. Phosphorylated EutV is postulated to disrupt terminator sites located just upstream of each of the genes eutP, eutG, eutS and eutA (Figure 2A); its association is therefore predicted to activate downstream gene expression [15]–[17]. These locations within the eut operon were found to share a common 13-nucleotide sequence (AGCAANGRRGCUY) overlapping the 5′-proximal portion of their corresponding intrinsic terminator elements. We previously proposed that these sites could serve as part of the recognition sequence for ANTAR-based regulators in order to promote antitermination and allow production of the downstream transcript [17]. Recent work investigating the regulation of eutG in E. faecalis supports the model that antitermination occurs at this consensus sequence [18]. However, no functional studies have yet identified the sequence or structural features that are specifically important for antitermination in the eut system or any other system that utilizes ANTAR-based regulatory proteins.

Bottom Line: The novel antiterminator structure consists of two small hairpins with highly conserved terminal loop residues, both features being essential for successful antitermination.Despite the unrelatedness of the species in which they are found, the majority of the ANTAR-associated genes are thematically related to nitrogen management.These data suggest that the central tenets for gene regulation by ANTAR antitermination occur widely in nature to specifically control nitrogen metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.

ABSTRACT
ANTAR proteins are widespread bacterial regulatory proteins that have RNA-binding output domains and utilize antitermination to control gene expression at the post-initiation level. An ANTAR protein, EutV, regulates the ethanolamine-utilization genes (eut) in Enterococcus faecalis. Using this system, we present genetic and biochemical evidence of a general mechanism of antitermination used by ANTARs, including details of the antiterminator structure. The novel antiterminator structure consists of two small hairpins with highly conserved terminal loop residues, both features being essential for successful antitermination. The ANTAR protein dimerizes and associates with its substrate RNA in response to signal-induced phosphorylation. Furthermore, bioinformatic searches using this conserved antiterminator motif identified many new ANTAR target RNAs in phylogenetically diverse bacterial species, some comprising complex regulons. Despite the unrelatedness of the species in which they are found, the majority of the ANTAR-associated genes are thematically related to nitrogen management. These data suggest that the central tenets for gene regulation by ANTAR antitermination occur widely in nature to specifically control nitrogen metabolism.

Show MeSH