The tertiary motifs in complex RNA molecules play vital roles to either stabilize the formation of RNA 3D structure or to provide important biological functionality to the molecule. In order to better understand the roles of these tertiary motifs in riboswitches, we examined 11 representative riboswitch PDB structures for potential agreement of both motif occurrences and conservations. A total of 61 unique tertiary interactions were found in the reference structures. In addition to the expected common A-minor motifs and base-triples mainly involved in linking distant regions the riboswitch structures three highly conserved variants of A-minor interactions called G-minors were found in the SAM-I and FMN riboswitches where they appear to be involved in the recognition of the respective ligand's functional groups. From our structural survey as well as corresponding structure and sequence alignments, the agreement between motif occurrences and conservations are very prominent across the representative riboswitches. Our analysis provide evidence that some of these tertiary interactions are essential components to form the structure where their sequence positions are conserved despite a high degree of diversity in other parts of the respective riboswitches sequences. This is indicative of a vital role for these tertiary interactions in determining the specific biological function of riboswitch.
A major component of RNA structure stabilization are the hydrogen bonded interactions between the base residues. The importance and biological relevance for large clusters of base interactions can be much more easily investigated when their occurrences have been systematically detected, catalogued and compared. In this paper, we describe the database InterRNA (INTERactions in RNA structures database-http://mfrlab.org/interrna/) that contains records of known RNA 3D motifs as well as records for clusters of bases that are interconnected by hydrogen bonds. The contents of the database were compiled from RNA structural annotations carried out by the NASSAM (http://mfrlab.org/grafss/nassam) and COGNAC (http://mfrlab.org/grafss/cognac) computer programs. An analysis of the database content and comparisons with the existing corpus of knowledge regarding RNA 3D motifs clearly show that InterRNA is able to provide an extension of the annotations for known motifs as well as able to provide novel interactions for further investigations.
Similarities in the 3D patterns of RNA base interactions or arrangements can provide insights into their functions and roles in stabilization of the RNA 3D structure. Nucleic Acids Search for Substructures and Motifs (NASSAM) is a graph theoretical program that can search for 3D patterns of base arrangements by representing the bases as pseudo-atoms. The geometric relationship of the pseudo-atoms to each other as a pattern can be represented as a labeled graph where the pseudo-atoms are the graph's nodes while the edges are the inter-pseudo-atomic distances. The input files for NASSAM are PDB formatted 3D coordinates. This web server can be used to identify matches of base arrangement patterns in a query structure to annotated patterns that have been reported in the literature or that have possible functional and structural stabilization implications. The NASSAM program is freely accessible without any login requirement at http://mfrlab.org/grafss/nassam/.
The glycine riboswitch is a known regulatory element that is unique in having two aptamers that are joined by a linker region. In this study, we investigated a glycine riboswitch located in the 5' untranslated region of a glycine cleavage system homolog (gcvTHP) in Burkholderia spp. Structure prediction using the sequence generated a model with a glycine binding pocket composed of base-triple interactions (G62-A64-A86 and G65-U84-C85) that are supported by A/G minor interactions (A17-C60-G88 and G16-C61-G87, respectively) and two ribose-zipper motifs (C11-G12 interacting with A248-A247 and C153-U154 interacting with A79-A78) which had not been previously reported. The capacity of the riboswitch to bind to glycine was experimentally validated by native gel assays and the crucial role of interactions that make up the glycine binding pocket were proven by mutations of A17U and G16C which resulted in conformational differences that may lead to dysfunction. Using glycine supplemented minimal media, we were able to prove that the expression of the gcvTHP genes found downstream of the riboswitch responded to the glycine concentrations introduced thus confirming the role of this highly conserved Burkholderia riboswitch and its associated genes as a putative glycine detoxification system in Burkholderia spp.