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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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Related in: MedlinePlus

Phosphorylation of EutV correlates with improved RNA-binding affinity.Beryllium fluoride was added to EutV to mimic phosphorylated protein. To measure binding activity using an experimental method that was unaffected by beryllium fluoride we employed the differential radial capillary action of ligand assay (DRaCALA) [25]–[26]. A) For this experiment, a 5′-radiolabeled RNA was incubated with increasing amounts of protein and spotted onto a nitrocellulose membrane. Proteins bound to ligands are sequestered to the membrane at an inner spot, whereas all unbound ligand is able to radially diffuse away from the spot by capillary action. Using this assay, the RNA-binding activity of EutV was tested for the wild-type dual hairpin RNA or a mutant RNA containing oligo-uridines in the terminal loops. These reactions were conducted in the presence and absence of beryllium fluoride. B) DRaCALA data was plotted as the fraction bound versus EutV concentration. EutV in the presence of beryllium fluoride binds the wild-type dual hairpin RNA (green) with a higher affinity than in the absence of beryllium fluoride (red). Competition with unlabeled wild-type RNA (blue) at 10-fold excess reduced the fraction of bound RNA to the level seen in the absence of beryllium fluoride. EutV does not bind the mutant RNA in either the presence (open square) or absence (open triangle) of beryllium fluoride. C) A general model for the binding of RNA by EutV is presented herein, which is based on the aggregate data and is discussed in the text.
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pgen-1002666-g006: Phosphorylation of EutV correlates with improved RNA-binding affinity.Beryllium fluoride was added to EutV to mimic phosphorylated protein. To measure binding activity using an experimental method that was unaffected by beryllium fluoride we employed the differential radial capillary action of ligand assay (DRaCALA) [25]–[26]. A) For this experiment, a 5′-radiolabeled RNA was incubated with increasing amounts of protein and spotted onto a nitrocellulose membrane. Proteins bound to ligands are sequestered to the membrane at an inner spot, whereas all unbound ligand is able to radially diffuse away from the spot by capillary action. Using this assay, the RNA-binding activity of EutV was tested for the wild-type dual hairpin RNA or a mutant RNA containing oligo-uridines in the terminal loops. These reactions were conducted in the presence and absence of beryllium fluoride. B) DRaCALA data was plotted as the fraction bound versus EutV concentration. EutV in the presence of beryllium fluoride binds the wild-type dual hairpin RNA (green) with a higher affinity than in the absence of beryllium fluoride (red). Competition with unlabeled wild-type RNA (blue) at 10-fold excess reduced the fraction of bound RNA to the level seen in the absence of beryllium fluoride. EutV does not bind the mutant RNA in either the presence (open square) or absence (open triangle) of beryllium fluoride. C) A general model for the binding of RNA by EutV is presented herein, which is based on the aggregate data and is discussed in the text.

Mentions: Many response regulators are capable of autophosphorylation in the presence of small molecule phospho-donors such as acetyl phosphate, carbamoyl phosphate, or phosphoramidate. We tested the two most common small-molecule phosphodonors (acetyl phosphate and carbamoyl phosphate) for their ability to induce dimer formation. Although EutV did not form dimers in response to addition of these small molecules, they appeared to provoke a moderate conformational change in EutV, visualized as a delay in the elution volume. However, further tests revealed that magnesium alone was responsible for promoting the moderate conformational change in EutV as it had also been included with the small molecule phosphodonor solutions (Figure 5C). Since the SEC-MALLS experiments suggested that small molecule phosphodonors were unable to promote dimerization we reasoned that they could not be used as tools for probing the effects of phosphorylation-induced dimerization on RNA-binding activity. For many response regulators, the half-lives of the phosphorylated receiver domains can be very short due to the intrinsically labile aspartyl phosphate bond [21]–[22]. As an alternative, we added beryllium fluoride as a nonhydrolyzable mimic of phospho-aspartate [23]–[24] and measured EutV RNA-binding activity. Preliminary experiments with addition of beryllium fluoride to EutV revealed that the beryllofluoride addition negatively affected resolution of the EutV-RNA complexes in the EMSA assay format (data not shown). Therefore, a recently developed non-electrophoresis method called differential radial capillary action of ligand assay (DRaCALA) was instead employed for these purposes [25]–[26]. DRaCALA is a rapid and quantitative assay for protein-ligand interactions that is based on the ability of nitrocellulose membranes to preferentially sequester proteins over small molecule or nucleic acid ligands. Specifically, proteins and their radiolabeled ligands are immobilized together when spotted onto nitrocellulose membranes, while unbound radiolabeled ligands freely diffuse by capillary action away from the protein spot. The fraction of the targeted protein bound with its mobile ligand can be easily calculated using this assay, which has been validated in recent publications for proteins that bind small molecules [25] and nucleic acids [26]. Using DRaCALA, we radiolabeled the two hairpin RNA motif and quantified binding to EutV in the presence or absence of beryllium fluoride (Figure 6A–6B). The binding affinity of unphoshorylated EutV for the two hairpin RNA motif as measured by DRaCALA was similar to that seen previously by EMSA, further validating the use of this method. Addition of beryllium fluoride provoked a significant increase in RNA-binding activity for wild-type RNA but not for a negative control RNA containing mutations in the terminal loops. Moreover, addition of cold competitor RNA restored the apparent fraction bound to background levels.


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)

Phosphorylation of EutV correlates with improved RNA-binding affinity.Beryllium fluoride was added to EutV to mimic phosphorylated protein. To measure binding activity using an experimental method that was unaffected by beryllium fluoride we employed the differential radial capillary action of ligand assay (DRaCALA) [25]–[26]. A) For this experiment, a 5′-radiolabeled RNA was incubated with increasing amounts of protein and spotted onto a nitrocellulose membrane. Proteins bound to ligands are sequestered to the membrane at an inner spot, whereas all unbound ligand is able to radially diffuse away from the spot by capillary action. Using this assay, the RNA-binding activity of EutV was tested for the wild-type dual hairpin RNA or a mutant RNA containing oligo-uridines in the terminal loops. These reactions were conducted in the presence and absence of beryllium fluoride. B) DRaCALA data was plotted as the fraction bound versus EutV concentration. EutV in the presence of beryllium fluoride binds the wild-type dual hairpin RNA (green) with a higher affinity than in the absence of beryllium fluoride (red). Competition with unlabeled wild-type RNA (blue) at 10-fold excess reduced the fraction of bound RNA to the level seen in the absence of beryllium fluoride. EutV does not bind the mutant RNA in either the presence (open square) or absence (open triangle) of beryllium fluoride. C) A general model for the binding of RNA by EutV is presented herein, which is based on the aggregate data and is discussed in the text.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002666-g006: Phosphorylation of EutV correlates with improved RNA-binding affinity.Beryllium fluoride was added to EutV to mimic phosphorylated protein. To measure binding activity using an experimental method that was unaffected by beryllium fluoride we employed the differential radial capillary action of ligand assay (DRaCALA) [25]–[26]. A) For this experiment, a 5′-radiolabeled RNA was incubated with increasing amounts of protein and spotted onto a nitrocellulose membrane. Proteins bound to ligands are sequestered to the membrane at an inner spot, whereas all unbound ligand is able to radially diffuse away from the spot by capillary action. Using this assay, the RNA-binding activity of EutV was tested for the wild-type dual hairpin RNA or a mutant RNA containing oligo-uridines in the terminal loops. These reactions were conducted in the presence and absence of beryllium fluoride. B) DRaCALA data was plotted as the fraction bound versus EutV concentration. EutV in the presence of beryllium fluoride binds the wild-type dual hairpin RNA (green) with a higher affinity than in the absence of beryllium fluoride (red). Competition with unlabeled wild-type RNA (blue) at 10-fold excess reduced the fraction of bound RNA to the level seen in the absence of beryllium fluoride. EutV does not bind the mutant RNA in either the presence (open square) or absence (open triangle) of beryllium fluoride. C) A general model for the binding of RNA by EutV is presented herein, which is based on the aggregate data and is discussed in the text.
Mentions: Many response regulators are capable of autophosphorylation in the presence of small molecule phospho-donors such as acetyl phosphate, carbamoyl phosphate, or phosphoramidate. We tested the two most common small-molecule phosphodonors (acetyl phosphate and carbamoyl phosphate) for their ability to induce dimer formation. Although EutV did not form dimers in response to addition of these small molecules, they appeared to provoke a moderate conformational change in EutV, visualized as a delay in the elution volume. However, further tests revealed that magnesium alone was responsible for promoting the moderate conformational change in EutV as it had also been included with the small molecule phosphodonor solutions (Figure 5C). Since the SEC-MALLS experiments suggested that small molecule phosphodonors were unable to promote dimerization we reasoned that they could not be used as tools for probing the effects of phosphorylation-induced dimerization on RNA-binding activity. For many response regulators, the half-lives of the phosphorylated receiver domains can be very short due to the intrinsically labile aspartyl phosphate bond [21]–[22]. As an alternative, we added beryllium fluoride as a nonhydrolyzable mimic of phospho-aspartate [23]–[24] and measured EutV RNA-binding activity. Preliminary experiments with addition of beryllium fluoride to EutV revealed that the beryllofluoride addition negatively affected resolution of the EutV-RNA complexes in the EMSA assay format (data not shown). Therefore, a recently developed non-electrophoresis method called differential radial capillary action of ligand assay (DRaCALA) was instead employed for these purposes [25]–[26]. DRaCALA is a rapid and quantitative assay for protein-ligand interactions that is based on the ability of nitrocellulose membranes to preferentially sequester proteins over small molecule or nucleic acid ligands. Specifically, proteins and their radiolabeled ligands are immobilized together when spotted onto nitrocellulose membranes, while unbound radiolabeled ligands freely diffuse by capillary action away from the protein spot. The fraction of the targeted protein bound with its mobile ligand can be easily calculated using this assay, which has been validated in recent publications for proteins that bind small molecules [25] and nucleic acids [26]. Using DRaCALA, we radiolabeled the two hairpin RNA motif and quantified binding to EutV in the presence or absence of beryllium fluoride (Figure 6A–6B). The binding affinity of unphoshorylated EutV for the two hairpin RNA motif as measured by DRaCALA was similar to that seen previously by EMSA, further validating the use of this method. Addition of beryllium fluoride provoked a significant increase in RNA-binding activity for wild-type RNA but not for a negative control RNA containing mutations in the terminal loops. Moreover, addition of cold competitor RNA restored the apparent fraction bound to background levels.

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