Regulatory context drives conservation of glycine riboswitch aptamers

Abstract

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.

Structural Insights into RNA Dimerization: Motifs, Interfaces and Functions

“…Further studies showed that singlet glycine riboswitches bind glycine with comparable affinities as the more common tandem version, but require a neighboring “ghost aptamer”, which is likely a degenerate, reduced version of the original partner aptamer that maintained the inter-aptamer contacts for structural stabilization. There are multiple proposals on why most glycine riboswitches have retained a tandem arrangement, as such an arrangement does not apparently enhance ligand binding (at least in vitro) [ 145 , 146 , 147 , 148 , 149 , 150 ]. Here, we describe one additional hypothesis.…”

A fluoride-responsive genetic circuit enables in vivo biofluorination in engineered Pseudomonas putida

“…Riboswitches have been adapted as biosensors, affording easy screening of high-producer microbial strains 27 . However, the widespread use of these regulatory devices is limited by their strong contextual effect on gene expression 28,29 . A fluoride ion (F − )responsive riboswitch (FRS) has been described in bacteria and archaea, where it regulates the expression of genes involved in ion detoxification-typically controlling F − transporters 30 .…”

Siblings or doppelgängers? Deciphering the evolution of structured cis-regulatory RNAs beyond homology

“…Yet, extensive biochemical analysis of the tandem OFF switch (regulating transport) from Vibrio cholerae showed that ligand binding by the second aptamer is more important [30]. A recent computational analysis of glycine aptamers suggested that most singlet glycine riboswitches may derive from these tandem riboswitches [31]. In this scenario, the 'ghost' aptamer associated with most singlet Heat maps of the consensus structures for the tandem and singlet glycine riboswitches regulating glycine cleavage metabolism (GCV) or glycine transport proteins (TP), with the glycine-binding pocket indicated (diamond), alignments used from [31].…”

“…A recent computational analysis of glycine aptamers suggested that most singlet glycine riboswitches may derive from these tandem riboswitches [31]. In this scenario, the 'ghost' aptamer associated with most singlet Heat maps of the consensus structures for the tandem and singlet glycine riboswitches regulating glycine cleavage metabolism (GCV) or glycine transport proteins (TP), with the glycine-binding pocket indicated (diamond), alignments used from [31]. (A) Aptamer 1 is more highly conserved in the tandem GCV riboswitch and type-1 singleton, while aptamer 2 degrades into a conserved ghost aptamer important for tertiary structure.…”

“…riboswitches is a degraded form of the less critical aptamer within a tandem aptamer pair. Singlet glycine riboswitches are thus divided into type-1 switches if the ghost aptamer follows the ligand-binding aptamer (and most frequently precede glycine cleavage operons) or type-2 switches if the ghost aptamer precedes it (and most frequently precede transporters) [31,32]. The regulated genes appear to have driven whether the first or second aptamer is conserved, as the other aptamer degrades to a stem-loop ghost aptamer [30].…”