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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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Bioinformatic analysis of the ANTAR domain and its two stem-loop RNA substrate.A) Using a covariance-based search approach (Infernal [28]), we identified additional occurrences of the ANTAR RNA substrate in bacteria that contained eut pathways. The comparative sequence alignment of these RNA sequences is shown in Figure S2 and described in Table S1. A scatter plot is shown for the resulting RNA hits, where each data point represents a different RNA hit. The hit scores for these sequences were plotted for two classes of microorganisms used in this search. Specifically, organisms that are predicted to encode for ANTAR domain proteins (see Table S1) have more RNA hits with higher scores than a control set of organisms that appear to lack any ANTAR domain proteins. Also, these hits were screened using TransTerm for the presence of an intrinsic terminator hairpin located immediately downstream of the P2 helix. Only a subset of the hits satisfied this important criterion. B) A consensus secondary structure was derived from this sequence alignment and is shown herein. C) The covariance-based search approach was then employed against 1902 bacterial genomes to search more broadly for putative ANTAR-based regulatory pathways. Again, a scatter plot is shown for the resulting hits, and for the subsequent screening of these hits for the presence of an overlapping downstream intrinsic terminator hairpin. See also Figures S1, S2 and Tables S1, S2 for more information on the covariance search results.
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pgen-1002666-g007: Bioinformatic analysis of the ANTAR domain and its two stem-loop RNA substrate.A) Using a covariance-based search approach (Infernal [28]), we identified additional occurrences of the ANTAR RNA substrate in bacteria that contained eut pathways. The comparative sequence alignment of these RNA sequences is shown in Figure S2 and described in Table S1. A scatter plot is shown for the resulting RNA hits, where each data point represents a different RNA hit. The hit scores for these sequences were plotted for two classes of microorganisms used in this search. Specifically, organisms that are predicted to encode for ANTAR domain proteins (see Table S1) have more RNA hits with higher scores than a control set of organisms that appear to lack any ANTAR domain proteins. Also, these hits were screened using TransTerm for the presence of an intrinsic terminator hairpin located immediately downstream of the P2 helix. Only a subset of the hits satisfied this important criterion. B) A consensus secondary structure was derived from this sequence alignment and is shown herein. C) The covariance-based search approach was then employed against 1902 bacterial genomes to search more broadly for putative ANTAR-based regulatory pathways. Again, a scatter plot is shown for the resulting hits, and for the subsequent screening of these hits for the presence of an overlapping downstream intrinsic terminator hairpin. See also Figures S1, S2 and Tables S1, S2 for more information on the covariance search results.

Mentions: Given the close sequence and structural similarity between the dual hairpin RNA motif in the three different characterized ANTAR systems (AmiR, NasR, EutV), we hypothesized that the RNA motif as identified herein might be generally representative of ANTAR substrates in other organisms. Also, the three previously characterized ANTAR regulatory systems each affected a single locus in their respective host organisms, and we reasoned that a subset of bacteria might instead incorporate multiple ANTAR-responsive RNA elements at disparate genomic locations for coordination of ANTAR-based regulons. To this end, we searched for additional occurrences of the putative ANTAR RNA substrate using a bioinformatics-based approach. Specifically, we used a covariance model-based approach [28] wherein a basic sequence alignment of a target RNA element, including certain secondary and primary sequence determinants, is used as input criteria for discovery of additional representatives from fully sequenced bacterial genomes. This method has been successfully employed for larger, structured RNAs such as riboswitches, and is also the underlying algorithm currently used by the Rfam database team to curate bacterial noncoding RNAs [29]. Therefore, a seed alignment was created based on the putative ANTAR RNA substrates (the dual hairpin element) from the eut loci of E. faecalis, Clostridium and Listeria species, as well as the corresponding RNA sequences for Klebsiella oxytoca nasF and Pseudomonas aeruginosa amiE, which are the target substrates for NasR and AmiR, respectively (Figure S1). This RNA element was defined as a dual hairpin motif with a minimum of three base-pairs in each stem and a variable linker region connecting the two stems. Sequence conservation in the loops, with an adenine at position 1 and a guanine at position 4 of each loop was maintained. Given the relatively small size of the motif and the small number of residues conserved at the primary sequence level, the first search was targeted against a narrowly defined genomic subset. We reasoned that this would allow us to fully examine the quality of our individual RNA hits. For this target analysis we searched against 83 bacterial genomes that were previously predicted [30] to specifically encode for a putative eut locus. Some eut loci are regulated by a DNA-binding regulator called EutR (e.g., Salmonella, Escherichia) whereas others, especially the Firmicutes, are regulated by a RNA-binding, ANTAR-containing homolog of EutV, as in E. faecalis[30]. Therefore, a subset of these genomes contains putative eut pathway homologues but lack any ANTAR-encoding genes, while other genomes contain both. As predicted, we recovered less RNA hits in genomes that lack ANTAR-encoding genes (Figure 7A). Another strength of the subset of genomes chosen for the initial analysis is that they include phylogenetically diverse species representative of many different evolutionary lineages.


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)

Bioinformatic analysis of the ANTAR domain and its two stem-loop RNA substrate.A) Using a covariance-based search approach (Infernal [28]), we identified additional occurrences of the ANTAR RNA substrate in bacteria that contained eut pathways. The comparative sequence alignment of these RNA sequences is shown in Figure S2 and described in Table S1. A scatter plot is shown for the resulting RNA hits, where each data point represents a different RNA hit. The hit scores for these sequences were plotted for two classes of microorganisms used in this search. Specifically, organisms that are predicted to encode for ANTAR domain proteins (see Table S1) have more RNA hits with higher scores than a control set of organisms that appear to lack any ANTAR domain proteins. Also, these hits were screened using TransTerm for the presence of an intrinsic terminator hairpin located immediately downstream of the P2 helix. Only a subset of the hits satisfied this important criterion. B) A consensus secondary structure was derived from this sequence alignment and is shown herein. C) The covariance-based search approach was then employed against 1902 bacterial genomes to search more broadly for putative ANTAR-based regulatory pathways. Again, a scatter plot is shown for the resulting hits, and for the subsequent screening of these hits for the presence of an overlapping downstream intrinsic terminator hairpin. See also Figures S1, S2 and Tables S1, S2 for more information on the covariance search results.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002666-g007: Bioinformatic analysis of the ANTAR domain and its two stem-loop RNA substrate.A) Using a covariance-based search approach (Infernal [28]), we identified additional occurrences of the ANTAR RNA substrate in bacteria that contained eut pathways. The comparative sequence alignment of these RNA sequences is shown in Figure S2 and described in Table S1. A scatter plot is shown for the resulting RNA hits, where each data point represents a different RNA hit. The hit scores for these sequences were plotted for two classes of microorganisms used in this search. Specifically, organisms that are predicted to encode for ANTAR domain proteins (see Table S1) have more RNA hits with higher scores than a control set of organisms that appear to lack any ANTAR domain proteins. Also, these hits were screened using TransTerm for the presence of an intrinsic terminator hairpin located immediately downstream of the P2 helix. Only a subset of the hits satisfied this important criterion. B) A consensus secondary structure was derived from this sequence alignment and is shown herein. C) The covariance-based search approach was then employed against 1902 bacterial genomes to search more broadly for putative ANTAR-based regulatory pathways. Again, a scatter plot is shown for the resulting hits, and for the subsequent screening of these hits for the presence of an overlapping downstream intrinsic terminator hairpin. See also Figures S1, S2 and Tables S1, S2 for more information on the covariance search results.
Mentions: Given the close sequence and structural similarity between the dual hairpin RNA motif in the three different characterized ANTAR systems (AmiR, NasR, EutV), we hypothesized that the RNA motif as identified herein might be generally representative of ANTAR substrates in other organisms. Also, the three previously characterized ANTAR regulatory systems each affected a single locus in their respective host organisms, and we reasoned that a subset of bacteria might instead incorporate multiple ANTAR-responsive RNA elements at disparate genomic locations for coordination of ANTAR-based regulons. To this end, we searched for additional occurrences of the putative ANTAR RNA substrate using a bioinformatics-based approach. Specifically, we used a covariance model-based approach [28] wherein a basic sequence alignment of a target RNA element, including certain secondary and primary sequence determinants, is used as input criteria for discovery of additional representatives from fully sequenced bacterial genomes. This method has been successfully employed for larger, structured RNAs such as riboswitches, and is also the underlying algorithm currently used by the Rfam database team to curate bacterial noncoding RNAs [29]. Therefore, a seed alignment was created based on the putative ANTAR RNA substrates (the dual hairpin element) from the eut loci of E. faecalis, Clostridium and Listeria species, as well as the corresponding RNA sequences for Klebsiella oxytoca nasF and Pseudomonas aeruginosa amiE, which are the target substrates for NasR and AmiR, respectively (Figure S1). This RNA element was defined as a dual hairpin motif with a minimum of three base-pairs in each stem and a variable linker region connecting the two stems. Sequence conservation in the loops, with an adenine at position 1 and a guanine at position 4 of each loop was maintained. Given the relatively small size of the motif and the small number of residues conserved at the primary sequence level, the first search was targeted against a narrowly defined genomic subset. We reasoned that this would allow us to fully examine the quality of our individual RNA hits. For this target analysis we searched against 83 bacterial genomes that were previously predicted [30] to specifically encode for a putative eut locus. Some eut loci are regulated by a DNA-binding regulator called EutR (e.g., Salmonella, Escherichia) whereas others, especially the Firmicutes, are regulated by a RNA-binding, ANTAR-containing homolog of EutV, as in E. faecalis[30]. Therefore, a subset of these genomes contains putative eut pathway homologues but lack any ANTAR-encoding genes, while other genomes contain both. As predicted, we recovered less RNA hits in genomes that lack ANTAR-encoding genes (Figure 7A). Another strength of the subset of genomes chosen for the initial analysis is that they include phylogenetically diverse species representative of many different evolutionary lineages.

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