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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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Responsiveness of the ef0120 leader region to regulation by EutVW.A) One outcome of the covariance searches was identification of a new putative ANTAR hit within the E. faecalis genome. This hit contained an overlapping intrinsic terminator hairpin, as identified by TransTermHP, despite the fact that a terminator was not a stringent requirement of the initial search criteria. B) To test whether this hit was functionally responsive to EutVW in vivo, the leader region of this gene was translationally fused to a lacZ reporter and monitored with and without AdoCbl and ethanolamine.
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pgen-1002666-g008: Responsiveness of the ef0120 leader region to regulation by EutVW.A) One outcome of the covariance searches was identification of a new putative ANTAR hit within the E. faecalis genome. This hit contained an overlapping intrinsic terminator hairpin, as identified by TransTermHP, despite the fact that a terminator was not a stringent requirement of the initial search criteria. B) To test whether this hit was functionally responsive to EutVW in vivo, the leader region of this gene was translationally fused to a lacZ reporter and monitored with and without AdoCbl and ethanolamine.

Mentions: Interestingly, while most of the new hits in this search were associated with eut genes, many were not. For example, a new ANTAR substrate was unexpectedly identified in the E. faecalis genome outside of the eut locus and within the 5′ leader region of ef0120, suggesting that EutVW might indeed control a regulon rather than a single locus. To investigate this observation further we fused the ef0120 5′ leader region to lacZ and monitored expression in the presence and absence of AdoCbl, ethanolamine, and the EutVW genes (Figure 8). Indeed, expression was activated by AdoCbl and ethanolamine in a EutVW-dependent manner. Therefore, our covariance search of putative ANTAR substrates is likely to have revealed ANTAR-based coordination of E. faecalis genes from both inside and outside of the eut locus. In fact, this search revealed many examples where putative ANTAR substrates were associated with multiple functionally related operons, as one might expect for regulons. For example, new ANTAR substrates appeared to be co-transcriptionally linked to different glutamate synthase genes in the Desulfotomaculum reducens genome (Figure 9A). Similarly, in Mycobacterium vanbaalenii, a putative ANTAR RNA substrate is positioned upstream of multiple uncharacterized gene clusters unrelated to eut genes (data not shown). In Pelobacter carbinolicus new ANTAR substrates were located within three separate transcriptional units, which are each predicted to be important for nitrogenase function, suggesting a comprehensive ANTAR-based regulon for nitrogenase regulation in this microorganism (Figure 9B).


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)

Responsiveness of the ef0120 leader region to regulation by EutVW.A) One outcome of the covariance searches was identification of a new putative ANTAR hit within the E. faecalis genome. This hit contained an overlapping intrinsic terminator hairpin, as identified by TransTermHP, despite the fact that a terminator was not a stringent requirement of the initial search criteria. B) To test whether this hit was functionally responsive to EutVW in vivo, the leader region of this gene was translationally fused to a lacZ reporter and monitored with and without AdoCbl and ethanolamine.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002666-g008: Responsiveness of the ef0120 leader region to regulation by EutVW.A) One outcome of the covariance searches was identification of a new putative ANTAR hit within the E. faecalis genome. This hit contained an overlapping intrinsic terminator hairpin, as identified by TransTermHP, despite the fact that a terminator was not a stringent requirement of the initial search criteria. B) To test whether this hit was functionally responsive to EutVW in vivo, the leader region of this gene was translationally fused to a lacZ reporter and monitored with and without AdoCbl and ethanolamine.
Mentions: Interestingly, while most of the new hits in this search were associated with eut genes, many were not. For example, a new ANTAR substrate was unexpectedly identified in the E. faecalis genome outside of the eut locus and within the 5′ leader region of ef0120, suggesting that EutVW might indeed control a regulon rather than a single locus. To investigate this observation further we fused the ef0120 5′ leader region to lacZ and monitored expression in the presence and absence of AdoCbl, ethanolamine, and the EutVW genes (Figure 8). Indeed, expression was activated by AdoCbl and ethanolamine in a EutVW-dependent manner. Therefore, our covariance search of putative ANTAR substrates is likely to have revealed ANTAR-based coordination of E. faecalis genes from both inside and outside of the eut locus. In fact, this search revealed many examples where putative ANTAR substrates were associated with multiple functionally related operons, as one might expect for regulons. For example, new ANTAR substrates appeared to be co-transcriptionally linked to different glutamate synthase genes in the Desulfotomaculum reducens genome (Figure 9A). Similarly, in Mycobacterium vanbaalenii, a putative ANTAR RNA substrate is positioned upstream of multiple uncharacterized gene clusters unrelated to eut genes (data not shown). In Pelobacter carbinolicus new ANTAR substrates were located within three separate transcriptional units, which are each predicted to be important for nitrogenase function, suggesting a comprehensive ANTAR-based regulon for nitrogenase regulation in this microorganism (Figure 9B).

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