Characterization of an Orphan Riboswitch: Identification of a Metal-Sensing Regulatory RNA




Wakeman, Catherine Ann

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Riboswitches are RNA-based genetic control elements found in untranslated regions of the mRNA transcript that they regulate. These RNA motifs are highly structured and bind metabolites to elicit control of gene expression. Typically, the metabolite sensed by these RNAs is a component of the metabolic pathway in which the regulated gene product resides. The focus of this project has been the identification of the ligand for a riboswitch that was discovered using bioinformatics-based search methods. This riboswitch was designated the ykoK RNA element due to its location in the 5' UTR of the B. subtilis ykoK (mgtE) gene, which appears to be a magnesium transporter. Therefore, the possibility that this RNA senses magnesium levels was explored. The data revealed that the RNA element imparts magnesium-responsive regulation to the ykoK gene. These data also indicated which portions of the RNA are essential for genetic regulation. The results of a battery of biochemical tests demonstrated that magnesium triggers a concerted conformational change in the RNA such that it adopts a compacted tertiary structure. Resolution of the three-dimensional structure of the RNA in the magnesium bound state revealed the basis of this metal-induced tertiary conformation and how this relates to genetic control. Intriguingly, this structure revealed the presence of six magnesium ions, making this the first example of multiple ligands binding to a single riboswitch aptamer. When individual metal-binding sites were eliminated using phosphorothioate substitutions, it became evident that all six of these magnesium-binding sites and up to three additional metal-binding sites are required for function of this RNA. Therefore, these data demonstrate that the ykoK RNA element, now designated the M-box RNA, directly senses intracellular magnesium levels for the purposes of genetic control. These findings should have broad implications given that this RNA element is wide spread among Gram-positive bacteria and appears to regulate many additional gene categories such as ABC transporters, cell division proteins, and proteins of unknown function. The exploration of the connection between magnesium concentration and the expression levels of these proteins might provide insights into previously undefined functional roles.

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