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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 EutV ANTAR regulator specifically recognizes a dual hairpin RNA motif.A) Alternate structures formed by the conserved RNA motif are shown. L1 and L2 denote the two terminal loops identified herein and predicted to be involved in ANTAR recognition (green). Altered base pairing allows formation of an alternate RNA structure that includes a terminator stem-loop (red). B) The primary sequence and secondary structure is shown for the dual hairpin RNA motif from the eutP 5′ leader region. Numbering reflects the transcription start site as +1. C) In vivo analysis of RNA mutants using lacZ reporter fusions to the eutP 5′ leader region is summarized as bar graphs. Deletion of eutVW (yellow) abolished induction of lacZ as compared to the wt (red). Deletion of the P1 stem-loop (green) or a mutation in the P2 stem-loop (grey) also negatively affected eutP inducibility. Mutation of the first (adenine) or fourth (guanine) nucleotide within the hexanucleotide loops L1 (blue stripes, yellow stripes) and L2 (grey stripes, pink stripes) significantly decreased eutP induction. Mutations affecting the P1 stem structure (brown) decreased induction but induction could be regained with compensatory mutations that restored the stem structure (blue). Mutations in the sequence of the closing base-pairs of stem-loop P1 abolished induction, even while maintaining the secondary structure (light blue). An increase or decrease in the length of the linker separating P1 and P2 (orange, light green) negatively affected induction. D) Binding isotherms derived from electrophoretic mobility shift assays (EMSA) are shown. Fractional saturation is plotted against protein concentration. EutV (unphosphorylated) bound the eutP 5′ leader region with an apparent KD of 10 µM (black). Binding was significantly deceased in an RNA mutant where the hexanucleotide terminal loops were mutated to uridines (grey). Binding was significantly weaker with RNAs mutated in the first (red) and fourth (open circle) positions of the terminal loops. See also Figure S1 for more information on the seed alignment that was used to derive the predicted RNA secondary structure and Figure S4 for information on EutV purification.
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pgen-1002666-g003: The EutV ANTAR regulator specifically recognizes a dual hairpin RNA motif.A) Alternate structures formed by the conserved RNA motif are shown. L1 and L2 denote the two terminal loops identified herein and predicted to be involved in ANTAR recognition (green). Altered base pairing allows formation of an alternate RNA structure that includes a terminator stem-loop (red). B) The primary sequence and secondary structure is shown for the dual hairpin RNA motif from the eutP 5′ leader region. Numbering reflects the transcription start site as +1. C) In vivo analysis of RNA mutants using lacZ reporter fusions to the eutP 5′ leader region is summarized as bar graphs. Deletion of eutVW (yellow) abolished induction of lacZ as compared to the wt (red). Deletion of the P1 stem-loop (green) or a mutation in the P2 stem-loop (grey) also negatively affected eutP inducibility. Mutation of the first (adenine) or fourth (guanine) nucleotide within the hexanucleotide loops L1 (blue stripes, yellow stripes) and L2 (grey stripes, pink stripes) significantly decreased eutP induction. Mutations affecting the P1 stem structure (brown) decreased induction but induction could be regained with compensatory mutations that restored the stem structure (blue). Mutations in the sequence of the closing base-pairs of stem-loop P1 abolished induction, even while maintaining the secondary structure (light blue). An increase or decrease in the length of the linker separating P1 and P2 (orange, light green) negatively affected induction. D) Binding isotherms derived from electrophoretic mobility shift assays (EMSA) are shown. Fractional saturation is plotted against protein concentration. EutV (unphosphorylated) bound the eutP 5′ leader region with an apparent KD of 10 µM (black). Binding was significantly deceased in an RNA mutant where the hexanucleotide terminal loops were mutated to uridines (grey). Binding was significantly weaker with RNAs mutated in the first (red) and fourth (open circle) positions of the terminal loops. See also Figure S1 for more information on the seed alignment that was used to derive the predicted RNA secondary structure and Figure S4 for information on EutV purification.

Mentions: In total, the regions immediately upstream of eutP, eutG, eutS, and eutA are predicted to contain intrinsic terminators consisting of a stem-loop followed by a run of uridines (Figure 2A, Figure 3B). Deletion of these terminator elements from the eutP-lacZ and eutS-lacZ constructs resulted in high levels of unregulated expression (Figure 2B and 2C). These data, along with a recent investigation of the transcriptional terminator in the region upstream of eutG[18], validate a model in which the eut locus is regulated in part by a series of intrinsic transcriptional terminators interspersed throughout the operon. These terminator elements are postulated to keep gene expression off under non-inducing conditions. To increase downstream gene expression under inducing conditions, the model predicts that activated EutV prevents formation of these terminators.


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 EutV ANTAR regulator specifically recognizes a dual hairpin RNA motif.A) Alternate structures formed by the conserved RNA motif are shown. L1 and L2 denote the two terminal loops identified herein and predicted to be involved in ANTAR recognition (green). Altered base pairing allows formation of an alternate RNA structure that includes a terminator stem-loop (red). B) The primary sequence and secondary structure is shown for the dual hairpin RNA motif from the eutP 5′ leader region. Numbering reflects the transcription start site as +1. C) In vivo analysis of RNA mutants using lacZ reporter fusions to the eutP 5′ leader region is summarized as bar graphs. Deletion of eutVW (yellow) abolished induction of lacZ as compared to the wt (red). Deletion of the P1 stem-loop (green) or a mutation in the P2 stem-loop (grey) also negatively affected eutP inducibility. Mutation of the first (adenine) or fourth (guanine) nucleotide within the hexanucleotide loops L1 (blue stripes, yellow stripes) and L2 (grey stripes, pink stripes) significantly decreased eutP induction. Mutations affecting the P1 stem structure (brown) decreased induction but induction could be regained with compensatory mutations that restored the stem structure (blue). Mutations in the sequence of the closing base-pairs of stem-loop P1 abolished induction, even while maintaining the secondary structure (light blue). An increase or decrease in the length of the linker separating P1 and P2 (orange, light green) negatively affected induction. D) Binding isotherms derived from electrophoretic mobility shift assays (EMSA) are shown. Fractional saturation is plotted against protein concentration. EutV (unphosphorylated) bound the eutP 5′ leader region with an apparent KD of 10 µM (black). Binding was significantly deceased in an RNA mutant where the hexanucleotide terminal loops were mutated to uridines (grey). Binding was significantly weaker with RNAs mutated in the first (red) and fourth (open circle) positions of the terminal loops. See also Figure S1 for more information on the seed alignment that was used to derive the predicted RNA secondary structure and Figure S4 for information on EutV purification.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002666-g003: The EutV ANTAR regulator specifically recognizes a dual hairpin RNA motif.A) Alternate structures formed by the conserved RNA motif are shown. L1 and L2 denote the two terminal loops identified herein and predicted to be involved in ANTAR recognition (green). Altered base pairing allows formation of an alternate RNA structure that includes a terminator stem-loop (red). B) The primary sequence and secondary structure is shown for the dual hairpin RNA motif from the eutP 5′ leader region. Numbering reflects the transcription start site as +1. C) In vivo analysis of RNA mutants using lacZ reporter fusions to the eutP 5′ leader region is summarized as bar graphs. Deletion of eutVW (yellow) abolished induction of lacZ as compared to the wt (red). Deletion of the P1 stem-loop (green) or a mutation in the P2 stem-loop (grey) also negatively affected eutP inducibility. Mutation of the first (adenine) or fourth (guanine) nucleotide within the hexanucleotide loops L1 (blue stripes, yellow stripes) and L2 (grey stripes, pink stripes) significantly decreased eutP induction. Mutations affecting the P1 stem structure (brown) decreased induction but induction could be regained with compensatory mutations that restored the stem structure (blue). Mutations in the sequence of the closing base-pairs of stem-loop P1 abolished induction, even while maintaining the secondary structure (light blue). An increase or decrease in the length of the linker separating P1 and P2 (orange, light green) negatively affected induction. D) Binding isotherms derived from electrophoretic mobility shift assays (EMSA) are shown. Fractional saturation is plotted against protein concentration. EutV (unphosphorylated) bound the eutP 5′ leader region with an apparent KD of 10 µM (black). Binding was significantly deceased in an RNA mutant where the hexanucleotide terminal loops were mutated to uridines (grey). Binding was significantly weaker with RNAs mutated in the first (red) and fourth (open circle) positions of the terminal loops. See also Figure S1 for more information on the seed alignment that was used to derive the predicted RNA secondary structure and Figure S4 for information on EutV purification.
Mentions: In total, the regions immediately upstream of eutP, eutG, eutS, and eutA are predicted to contain intrinsic terminators consisting of a stem-loop followed by a run of uridines (Figure 2A, Figure 3B). Deletion of these terminator elements from the eutP-lacZ and eutS-lacZ constructs resulted in high levels of unregulated expression (Figure 2B and 2C). These data, along with a recent investigation of the transcriptional terminator in the region upstream of eutG[18], validate a model in which the eut locus is regulated in part by a series of intrinsic transcriptional terminators interspersed throughout the operon. These terminator elements are postulated to keep gene expression off under non-inducing conditions. To increase downstream gene expression under inducing conditions, the model predicts that activated EutV prevents formation of these terminators.

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