Nearly all representatives of experimentally validated riboswitch classes in bacteria control the expression of genes for the transport or synthesis of key metabolic compounds. Recent findings have revealed that some riboswitches also regulate genes involved in physiological changes, virulence, and stress responses. Many novel RNA motifs are being identified by using bioinformatics algorithms that search for conserved sequence and structural features located in intergenic regions. Some of these RNAs are likely to function as riboswitches for metabolites or signaling compounds, and confirmation of this function would reveal the basis of the genetic control of new regulons. Herein we describe the analysis of the ydaO riboswitch candidate, which represents one of the most widespread candidates remaining to be validated. These RNAs are common in Gram-positive bacteria, and their genomic associations with diverse genes suggest that they sense a compound that signals broader physiological changes. We determined that the ydaO motif exhibits sequence- and structure-dependent gene control, and reporter assays indicate that its natural ligand is present even when cells are grown in defined media. A transposon-mediated knockout screen resulted in mutants with a dysregulated expression of genes controlled by the RNA motif. The mutations disrupt genes that drastically modulate energy-generating pathways, suggesting that the intracellular concentration of the ligand sensed by the ydaO motif is altered under these stress conditions
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