It all comes together: examining structural elements of the glmS ribozyme in T. thermophilus
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Date
2017-04-17
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Johns Hopkins University
Abstract
The discovery of catalytic RNA shifted the traditional view of RNA as a passive messenger to one in which RNA occupied a regulatory role in cellular processes. Self- cleaving ribozymes and metabolite sensing riboswitches emerged as widespread and evolutionarily conserved mechanisms of controlling gene expression. One of these metabolite binding ribozymes resides in the 5’ UTR of an essential gene, glmS which encodes an enzyme involved in cell biosynthesis. A downstream metabolic product of glmS synthase, glucosamine-N-6- phosphate (GlcN6P), binds the glmS ribozyme resulting in self-cleavage of the 5’ leader sequence and degradation of the mRNA transcript. Thus, the glmS ribozyme acts as the key component in a negative feedback loop, tightly regulating expression of GlmS synthase. Here I investigated the significance of an additional module within the glmS ribozyme in a Gram-negative thermophilic bacterium, Thermus. thermophilus. This additional module contains a series of structures with RNA tertiary interaction motifs, which increase the stability of the RNA. Given the high temperature environment in which T. thermophilus resides, the presence of additional RNA tertiary motifs in a highly-conserved ribozyme like glmS suggests that the additional module is necessary for function. My data indicate that mutations to tertiary interactions within the additional module disrupt proper folding, leading to a loss of stability and a decrease in function. Rather than stabilize the core of the ribozyme at high temperature, I found that the additional module assists in folding the ribozyme into a functional state. A deeper understanding of the glmS ribozymes folded structure enhances our understanding of the active site of the ribozyme and the mechanism of ligand
binding. A GlcN6P analog that could trigger self-cleavage would result in inhibition of the essential gene GlmS synthase. Such an analog would therefore have potential as a broad-spectrum antibiotic. Development of such a drug requires detailed knowledge of the ribozymes folded structure, and this work contributes toward that task.
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glmS, ribozyme, thermophilic, RNA structure