Limits...
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.

Show MeSH

Related in: MedlinePlus

Dimerization of ANTAR-containing proteins.A) Comparison of size exclusion chromatography profiles (Superdex-75 column) for ANTARcc (blue), ANTAR (green) and protein calibration standards (grey) suggests a dimeric state for ANTARcc and ANTAR. The standard plot is shown as inset. B) Multi–angle laser light scattering (MALLS) data collected in tandem with size exclusion chromatography on a Superdex-200 column. MALLS analysis indicated that incubation of EutV with EutW in the absence of phosphorylating conditions (grey) did not result in a change in molar mass, whereas there was an altered elution time (marked as peak shift) as well as a change in molar mass from monomer to dimer upon incubation of EutV with EutW under phosphorylating conditions (red). C) A moderate change in elution volume (peak shift) occurred for EutV monomer (blue line) upon incubation with magnesium (orange), although this altered migration was not accompanied by an increase in the molar mass. Similarly, incubation of EutV with small-molecule phosphodonors acetyl phosphate (black) and carbamoyl phosphate (green) did not alter the monomeric state of EutV, beyond the shift in the peaks already attributed to magnesium addition. D) The theoretical molar masses for monomers of EutV protein variants are listed alongside experimentally observed values. SEC refers to size-exclusion chromatography and SEC-MALLS denotes size exclusion chromatography when coupled in tandem to detection by multi-angle laser light scattering.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3369931&req=5

pgen-1002666-g005: Dimerization of ANTAR-containing proteins.A) Comparison of size exclusion chromatography profiles (Superdex-75 column) for ANTARcc (blue), ANTAR (green) and protein calibration standards (grey) suggests a dimeric state for ANTARcc and ANTAR. The standard plot is shown as inset. B) Multi–angle laser light scattering (MALLS) data collected in tandem with size exclusion chromatography on a Superdex-200 column. MALLS analysis indicated that incubation of EutV with EutW in the absence of phosphorylating conditions (grey) did not result in a change in molar mass, whereas there was an altered elution time (marked as peak shift) as well as a change in molar mass from monomer to dimer upon incubation of EutV with EutW under phosphorylating conditions (red). C) A moderate change in elution volume (peak shift) occurred for EutV monomer (blue line) upon incubation with magnesium (orange), although this altered migration was not accompanied by an increase in the molar mass. Similarly, incubation of EutV with small-molecule phosphodonors acetyl phosphate (black) and carbamoyl phosphate (green) did not alter the monomeric state of EutV, beyond the shift in the peaks already attributed to magnesium addition. D) The theoretical molar masses for monomers of EutV protein variants are listed alongside experimentally observed values. SEC refers to size-exclusion chromatography and SEC-MALLS denotes size exclusion chromatography when coupled in tandem to detection by multi-angle laser light scattering.

Mentions: Bacterial response regulators often display the ability to form dimers or higher oligomers [12]. We speculated that in order to bind an RNA target that presents two similar surfaces for interaction, the protein component is also likely to form a dimer or higher ordered oligomeric state to recognize the RNA substrate. To test this, we first investigated the oligomeric state of the EutV ANTAR and ANTARcc domains using size-exclusion chromatography (SEC) (Figure 5A). While SEC is limited in the precise calculation of molar masses, the low extinction coefficients of these domains at 280 nm prevented the use of preferred techniques such as equilibrium analytical ultracentrifugation. From SEC, we inferred that both the ANTAR and ANTARcc domains formed dimers when compared to the elution profiles of the standard protein markers. Therefore, dimer formation appears to be an inherent characteristic of this domain, however, as discussed above and shown in Figure 4C, the presence of the coiled coil significantly improved the affinity for RNA-binding. This suggests that although both versions of the ANTAR domain are able to form dimers, there are likely to be differences between their dimeric conformations, that are crucial for RNA recognition.


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)

Dimerization of ANTAR-containing proteins.A) Comparison of size exclusion chromatography profiles (Superdex-75 column) for ANTARcc (blue), ANTAR (green) and protein calibration standards (grey) suggests a dimeric state for ANTARcc and ANTAR. The standard plot is shown as inset. B) Multi–angle laser light scattering (MALLS) data collected in tandem with size exclusion chromatography on a Superdex-200 column. MALLS analysis indicated that incubation of EutV with EutW in the absence of phosphorylating conditions (grey) did not result in a change in molar mass, whereas there was an altered elution time (marked as peak shift) as well as a change in molar mass from monomer to dimer upon incubation of EutV with EutW under phosphorylating conditions (red). C) A moderate change in elution volume (peak shift) occurred for EutV monomer (blue line) upon incubation with magnesium (orange), although this altered migration was not accompanied by an increase in the molar mass. Similarly, incubation of EutV with small-molecule phosphodonors acetyl phosphate (black) and carbamoyl phosphate (green) did not alter the monomeric state of EutV, beyond the shift in the peaks already attributed to magnesium addition. D) The theoretical molar masses for monomers of EutV protein variants are listed alongside experimentally observed values. SEC refers to size-exclusion chromatography and SEC-MALLS denotes size exclusion chromatography when coupled in tandem to detection by multi-angle laser light scattering.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002666-g005: Dimerization of ANTAR-containing proteins.A) Comparison of size exclusion chromatography profiles (Superdex-75 column) for ANTARcc (blue), ANTAR (green) and protein calibration standards (grey) suggests a dimeric state for ANTARcc and ANTAR. The standard plot is shown as inset. B) Multi–angle laser light scattering (MALLS) data collected in tandem with size exclusion chromatography on a Superdex-200 column. MALLS analysis indicated that incubation of EutV with EutW in the absence of phosphorylating conditions (grey) did not result in a change in molar mass, whereas there was an altered elution time (marked as peak shift) as well as a change in molar mass from monomer to dimer upon incubation of EutV with EutW under phosphorylating conditions (red). C) A moderate change in elution volume (peak shift) occurred for EutV monomer (blue line) upon incubation with magnesium (orange), although this altered migration was not accompanied by an increase in the molar mass. Similarly, incubation of EutV with small-molecule phosphodonors acetyl phosphate (black) and carbamoyl phosphate (green) did not alter the monomeric state of EutV, beyond the shift in the peaks already attributed to magnesium addition. D) The theoretical molar masses for monomers of EutV protein variants are listed alongside experimentally observed values. SEC refers to size-exclusion chromatography and SEC-MALLS denotes size exclusion chromatography when coupled in tandem to detection by multi-angle laser light scattering.
Mentions: Bacterial response regulators often display the ability to form dimers or higher oligomers [12]. We speculated that in order to bind an RNA target that presents two similar surfaces for interaction, the protein component is also likely to form a dimer or higher ordered oligomeric state to recognize the RNA substrate. To test this, we first investigated the oligomeric state of the EutV ANTAR and ANTARcc domains using size-exclusion chromatography (SEC) (Figure 5A). While SEC is limited in the precise calculation of molar masses, the low extinction coefficients of these domains at 280 nm prevented the use of preferred techniques such as equilibrium analytical ultracentrifugation. From SEC, we inferred that both the ANTAR and ANTARcc domains formed dimers when compared to the elution profiles of the standard protein markers. Therefore, dimer formation appears to be an inherent characteristic of this domain, however, as discussed above and shown in Figure 4C, the presence of the coiled coil significantly improved the affinity for RNA-binding. This suggests that although both versions of the ANTAR domain are able to form dimers, there are likely to be differences between their dimeric conformations, that are crucial for RNA recognition.

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
Related in: MedlinePlus