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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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The minimum ANTAR domain for RNA recognition.A) Three variants of EutV protein were investigated: (1) full length, which included the N-terminal receiver domain, a coiled coil region and the C-terminal ANTAR domain, (2) ANTARcc, which included the coiled coil region and the ANTAR domain, and (3) just the C-terminal ANTAR domain. B) A representative EMSA is shown for association of ANTARcc with 5′-radiolabeled RNA (eutP 5′ leader region) on a non-denaturing polyacrylamide gel. C) Binding isotherms derived from EMSAs showing fractional saturation versus protein concentration. These data suggested that the two-stem RNA motif is bound by ANTARcc (blue), full length EutV (black), and ANTAR (green) with decreasing affinity, respectively. D) ANTARcc binds different RNA constructs with variable affinities. While RNA that included the dual hairpin motif (blue) was bound with micromolar affinity, mutation of the terminal loop sequences (grey) as well as deletion of the second stem loop (squares) abolished binding. The sequences for the constructs are shown below.
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pgen-1002666-g004: The minimum ANTAR domain for RNA recognition.A) Three variants of EutV protein were investigated: (1) full length, which included the N-terminal receiver domain, a coiled coil region and the C-terminal ANTAR domain, (2) ANTARcc, which included the coiled coil region and the ANTAR domain, and (3) just the C-terminal ANTAR domain. B) A representative EMSA is shown for association of ANTARcc with 5′-radiolabeled RNA (eutP 5′ leader region) on a non-denaturing polyacrylamide gel. C) Binding isotherms derived from EMSAs showing fractional saturation versus protein concentration. These data suggested that the two-stem RNA motif is bound by ANTARcc (blue), full length EutV (black), and ANTAR (green) with decreasing affinity, respectively. D) ANTARcc binds different RNA constructs with variable affinities. While RNA that included the dual hairpin motif (blue) was bound with micromolar affinity, mutation of the terminal loop sequences (grey) as well as deletion of the second stem loop (squares) abolished binding. The sequences for the constructs are shown below.

Mentions: E. faecalis EutV is predicted to possess two domains - an N-terminal phospho-accepting receiver domain and a C-terminal ANTAR domain (Figure 4A). A region separating the two domains forms a coiled-coil as suggested by the COILS [19] server and by structural studies on AmiR as well as Rv1626, orthologs of EutV from Pseudomonas aeruginosa and Mycobacterium tuberculosis[5], [20]. The AmiR structure reveals an intimate dimer with an extended coiled-coil region, although the importance of the coiled coil region for the function of AmiR has not been studied. ANTAR itself is a poorly understood protein domain and little is known about its RNA-binding properties. Having identified the RNA target of the EutV ANTAR domain, we then investigated the protein domain requirements for RNA recognition. We expressed and purified two variants of the EutV ANTAR domain, which both lacked the response regulator receiver domain. One variant is referred herein as ANTARcc (which includes the putative coiled coil region) while the other variant is called ANTAR (which lacks the coiled coil region) (Figure 4A). Via EMSA experimentation using 5′ radioactively labeled RNA substrate and purified protein we determined the binding affinities of different recombinant proteins (Figure 4B–4C). ANTARcc binds the dual hairpin RNA substrate with an apparent affinity of ∼700 nM, a value that is 100-fold tighter as compared to ANTAR alone. This data suggests that the coiled-coil region plays an important structural role in EutV-RNA interactions. Also, as described earlier, full-length EutV in its unphosphorylated state binds RNA with an affinity of 10 µM (Figure 3D, Figure 4C), 10-fold weaker than ANTARcc. This suggests that in the unphosphorylated state, the receiver domain of EutV may damper RNA-binding activity of the ANTARcc domain. Phosphorylation of the receiver domain is likely to be accompanied by structural reorganization, perhaps allowing the ANTARcc domain to adopt a conformation better suited for RNA-binding.


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)

The minimum ANTAR domain for RNA recognition.A) Three variants of EutV protein were investigated: (1) full length, which included the N-terminal receiver domain, a coiled coil region and the C-terminal ANTAR domain, (2) ANTARcc, which included the coiled coil region and the ANTAR domain, and (3) just the C-terminal ANTAR domain. B) A representative EMSA is shown for association of ANTARcc with 5′-radiolabeled RNA (eutP 5′ leader region) on a non-denaturing polyacrylamide gel. C) Binding isotherms derived from EMSAs showing fractional saturation versus protein concentration. These data suggested that the two-stem RNA motif is bound by ANTARcc (blue), full length EutV (black), and ANTAR (green) with decreasing affinity, respectively. D) ANTARcc binds different RNA constructs with variable affinities. While RNA that included the dual hairpin motif (blue) was bound with micromolar affinity, mutation of the terminal loop sequences (grey) as well as deletion of the second stem loop (squares) abolished binding. The sequences for the constructs are shown below.
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Related In: Results  -  Collection

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

pgen-1002666-g004: The minimum ANTAR domain for RNA recognition.A) Three variants of EutV protein were investigated: (1) full length, which included the N-terminal receiver domain, a coiled coil region and the C-terminal ANTAR domain, (2) ANTARcc, which included the coiled coil region and the ANTAR domain, and (3) just the C-terminal ANTAR domain. B) A representative EMSA is shown for association of ANTARcc with 5′-radiolabeled RNA (eutP 5′ leader region) on a non-denaturing polyacrylamide gel. C) Binding isotherms derived from EMSAs showing fractional saturation versus protein concentration. These data suggested that the two-stem RNA motif is bound by ANTARcc (blue), full length EutV (black), and ANTAR (green) with decreasing affinity, respectively. D) ANTARcc binds different RNA constructs with variable affinities. While RNA that included the dual hairpin motif (blue) was bound with micromolar affinity, mutation of the terminal loop sequences (grey) as well as deletion of the second stem loop (squares) abolished binding. The sequences for the constructs are shown below.
Mentions: E. faecalis EutV is predicted to possess two domains - an N-terminal phospho-accepting receiver domain and a C-terminal ANTAR domain (Figure 4A). A region separating the two domains forms a coiled-coil as suggested by the COILS [19] server and by structural studies on AmiR as well as Rv1626, orthologs of EutV from Pseudomonas aeruginosa and Mycobacterium tuberculosis[5], [20]. The AmiR structure reveals an intimate dimer with an extended coiled-coil region, although the importance of the coiled coil region for the function of AmiR has not been studied. ANTAR itself is a poorly understood protein domain and little is known about its RNA-binding properties. Having identified the RNA target of the EutV ANTAR domain, we then investigated the protein domain requirements for RNA recognition. We expressed and purified two variants of the EutV ANTAR domain, which both lacked the response regulator receiver domain. One variant is referred herein as ANTARcc (which includes the putative coiled coil region) while the other variant is called ANTAR (which lacks the coiled coil region) (Figure 4A). Via EMSA experimentation using 5′ radioactively labeled RNA substrate and purified protein we determined the binding affinities of different recombinant proteins (Figure 4B–4C). ANTARcc binds the dual hairpin RNA substrate with an apparent affinity of ∼700 nM, a value that is 100-fold tighter as compared to ANTAR alone. This data suggests that the coiled-coil region plays an important structural role in EutV-RNA interactions. Also, as described earlier, full-length EutV in its unphosphorylated state binds RNA with an affinity of 10 µM (Figure 3D, Figure 4C), 10-fold weaker than ANTARcc. This suggests that in the unphosphorylated state, the receiver domain of EutV may damper RNA-binding activity of the ANTARcc domain. Phosphorylation of the receiver domain is likely to be accompanied by structural reorganization, perhaps allowing the ANTARcc domain to adopt a conformation better suited for RNA-binding.

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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