A riboswitch selective for the queuosine precursor preQ1 contains an unusually small aptamer domain

Abstract

A previous bioinformatics-based search for riboswitches yielded several candidate motifs in eubacteria. One of these motifs commonly resides in the 5' untranslated regions of genes involved in the biosynthesis of queuosine (Q), a hypermodified nucleoside occupying the anticodon wobble position of certain transfer RNAs. Here we show that this structured RNA is part of a riboswitch selective for 7-aminomethyl-7-deazaguanine (preQ(1)), an intermediate in queuosine biosynthesis. Compared with other natural metabolite-binding RNAs, the preQ(1) aptamer appears to have a simple structure, consisting of a single stem-loop and a short tail sequence that together are formed from as few as 34 nucleotides. Despite its small size, this aptamer is highly selective for its cognate ligand in vitro and has an affinity for preQ(1) in the low nanomolar range. Relatively compact RNA structures can therefore serve effectively as metabolite receptors to regulate gene expression.

Structural features of metabolite-sensing riboswitches

“…In addition, examination of the FMN-and vitamin B12-bound aptamers allows speculation about the binding of these same ligands to their naturally occuring riboswitch counterparts. In vitro evolved aptamers tend to be much smaller than their natural counterparts with minimal motifs comprising ~30-50 nucleotides [61] whereas riboswitches tend to be ~70-170 nucleotides in length (compare Figure 4 and Figure 2) with the recently published preQ 1 -binding riboswitch minimal aptamer domain being an outlier at ~34 nucleotides [36,21].…”

“…This observation, coupled with their widespread phylogenetic distribution, argues that riboswitches have been evolutionarily maintained for several billion years, although considerably more research is required to fully establish these claims. To date, biochemical evidence for riboswitch function has been obtained for RNAs that respond to adenosylcobalamin [14,15], thiamine pyrophosphate (TPP) [16], flavin mononucleotide (FMN) [17,18], guanine [19], adenine [20], a precursor for queuine [21], lysine [22], glycine [23], glucosamine-6-phosphate (GlcN6P) [4], and S-adenosylmethionine (SAM) [24-26; reviewed in 27,12]. Given their ability to function as sensitive sentinels for intracellular metabolites in a protein-independent manner, these RNAs are likely to require exquisite structural sophistication.…”

“…In addition, examination of the FMN-and vitamin B12-bound aptamers allows speculation about the binding of these same ligands to their naturally occuring riboswitch counterparts. In vitro evolved aptamers tend to be much smaller than their natural counterparts with minimal motifs comprising ~30-50 nucleotides [61] whereas riboswitches tend to be ~70-170 nucleotides in length (compare Figure 4 and Figure 2) with the recently published preQ 1 -binding riboswitch minimal aptamer domain being an outlier at ~34 nucleotides [36,21].Both riboswitch aptamer domains and RNA aptamers from in vitro evolution employ common mechanisms to bind small organic molecules, including base pairing, aromatic stacking, and metal-dependent stabilization of electronegative moieties, but they differ in the extent of direct and global interactions required to achieve molecule binding. In general, artificial aptamers do not interact with all portions of the ligand whereas natural aptamers (aptamer domains from riboswitches) typically bury the ligand in a site created by tertiary structure elements.…”

Single-pass classification of all noncoding sequences in a bacterial genome using phylogenetic profiles

“…Some of the labeled probes yield additional high molecular weight signals. Sequence analysis suggests that SA0347 (metB) is regulated by a T-box riboswitch and SA0667 (queC) by a preQ1 type riboswitch (Griffiths-Jones et al 2005;Roth et al 2007). Therefore, rsaOB and rsaOC are short transcripts likely resulting from premature transcription arrests of putatively longer transcripts that are also observed.…”

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ1riboswitches inE.coli

“…Bacterial riboswitches responsive to preQ 1 are currently known to fall into three phylogenetically distinct classes. The preQ 1 -I (class 1) aptamer is distributed widely and rather compact, comprising not more than 34 nucleotides [28]. The preQ 1 -II (class 2) riboswitch is about twice this size and has been found in the Firmicutes [29].…”