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.

Riboswitches are intricate, metabolite-binding RNA structures found in the non-coding regions of mRNA. Allosteric changes in the riboswitch that are induced by metabolite binding are harnessed to control the genes of a variety of essential metabolic pathways in eubacteria and in some eukaryotes. In this chapter, we describe an RNA structure analysis technique called "in-line probing." This assay makes use of the natural instability of RNA to elucidate secondary structure characteristics and ligand-binding capabilities of riboswitches in an entirely protein-free manner. Although this method has most frequently been used to examine riboswitches, the structures of any RNAs can be examined using this technique.

SummaryWe have identified a highly conserved RNA motif located upstream of genes encoding molybdate transporters, molybdenum cofactor (Moco) biosynthesis enzymes, and proteins that utilize Moco as a coenzyme. Bioinformatics searches have identified 176 representatives in g-Proteobacteria, d-Proteobacteria, Clostridia, Actinobacteria, Deinococcus-Thermus species and DNAs from environmental samples. Using genetic assays, we demonstrate that a Moco RNA in Escherichia coli associated with the Moco biosynthetic operon controls gene expression in response to Moco production. In addition, we provide evidence indicating that this conserved RNA discriminates against closely related analogues of Moco. These results, together with extensive phylogenetic conservation and typical gene control structures near some examples, indicate that representatives of this structured RNA represent a novel class of riboswitches that sense Moco. Furthermore, we identify variants of this RNA that are likely to be triggered by the related tungsten cofactor (Tuco), which carries tungsten in place of molybdenum as the metal constituent.

A novel family of riboswitches, called SAM-IV, is the fourth distinct set of mRNA elements to be reported that regulate gene expression via direct sensing of S-adenosylmethionine (SAM or AdoMet). SAM-IV riboswitches share conserved nucleotide positions with the previously described SAM-I riboswitches, despite rearranged structures and nucleotide positions with familyspecific nucleotide identities. Sequence analysis and molecular recognition experiments suggest that SAM-I and SAM-IV riboswitches share similar ligand binding sites, but have different scaffolds. Our findings support the view that RNA has considerable structural versatility and reveal that riboswitches exploit this potential to expand the scope of RNA in genetic regulation.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen causing skin and soft tissue, respiratory, and bloodstream infections. The type III secretion system (T3SS) is one important virulence factor. Production of the T3SS is controlled by ExsA, a transcription factor that activates expression of the entire T3SS regulon. Global regulators including Vfr, RsmA, and Hfq also contribute to regulation of the T3SS. Vfr is a cAMP-responsive transcription factor that activates exsA transcription. RsmA, an RNA-binding protein, inversely controls expression of the T3SS and the type VI secretion system (T6SS). Hfq is an RNA chaperone that functions by stabilizing small noncoding RNAs (sRNAs) and/or facilitating base pairing between sRNAs and mRNA targets. A previous study identified sRNA 1061, which directly targets the exsA mRNA and likely inhibits ExsA synthesis. In this study, we screened an sRNA expression library and identified sRNA 179 as an Hfq-dependent inhibitor of T3SS gene expression. Further characterization revealed that sRNA 179 inhibits the synthesis of both ExsA and Vfr. The previous finding that RsmA stimulates ExsA and Vfr synthesis suggested that sRNA 179 impacts the Gac/Rsm system. Consistent with that idea, the inhibitory activity of sRNA 179 is suppressed in a mutant lacking rsmY and rsmZ, and sRNA 179 expression stimulates rsmY transcription. RsmY and RsmZ are small noncoding RNAs that sequester RsmA from target mRNAs. Our combined findings show that Hfq and sRNA 179 indirectly regulate ExsA and Vfr synthesis by reducing the available pool of RsmA, leading to reduced expression of the T3SS and cAMP-Vfr regulons. IMPORTANCE Control of gene expression by small noncoding RNA (sRNA) is well documented but underappreciated. Deep sequencing of mRNA preparations from Pseudomonas aeruginosa suggests that >500 sRNAs are generated. Few of those sRNAs have defined roles in gene expression. To address that knowledge gap, we constructed an sRNA expression library and identified sRNA 179 as a regulator of the type III secretion system (T3SS) and the cAMP-Vfr regulons. The T3SS- and cAMP-Vfr-controlled genes are critical virulence factors. Increased understanding of the signals and regulatory mechanisms that control these important factors will enhance our understanding of disease progression and reveal potential approaches for therapeutic intervention.