Regulatory context drives conservation of glycine riboswitch aptamers

In comparison to protein coding sequences, the impact of mutation and natural selection on the sequence and function of non-coding (ncRNA) genes is not well understood. Many ncRNA genes are narrowly distributed to only a few organisms, and appear to be rapidly evolving. Compared to protein coding sequences, there are many challenges associated with assessment of ncRNAs that are not well addressed by conventional phylogenetic approaches, including: short sequence length, lack of primary sequence conservation, and the importance of secondary structure for biological function. Riboswitches are structured ncRNAs that directly interact with small molecules to regulate gene expression in bacteria. They typically consist of a ligand-binding domain (aptamer) whose folding changes drive changes in gene expression. The glycine riboswitch is among the most well-studied due to the widespread occurrence of a tandem aptamer arrangement (tandem), wherein two homologous aptamers interact with glycine and each other to regulate gene expression. However, a significant proportion of glycine riboswitches are comprised of single aptamers (singleton). Here we use graph clustering to circumvent the limitations of traditional phylogenetic analysis when studying the relationship between the tandem and singleton glycine aptamers. Graph clustering enables a broader range of pairwise comparison measures to be used to assess aptamer similarity. Using this approach, we show that one aptamer of the tandem glycine riboswitch pair is typically much more highly conserved, and that which aptamer is conserved depends on the regulated gene. Furthermore, our analysis also reveals that singleton aptamers are more similar to either the first or second tandem aptamer, again based on the regulated gene. Taken together, our findings suggest that tandem glycine riboswitches degrade into functional singletons, with the regulated gene(s) dictating which glycine-binding aptamer is conserved.