Date of Award
Bachelor of Science
In most Gram-positive bacteria, including important pathogens, expression of many aminoacyl tRNA synthetase (aaRS) genes is controlled by the tRNA substrate specific to each of these enzymes. This riboswitch regulatory mechanism is unique to Gram-positive bacteria and because correct and efficient aminoacylation of tRNAs is essential to an organism’s viability, it is an ideal target for the development of new antibiotics. The 5’-untranslated region (5’UTR) of the aaRS mRNA adopts a conformation that determines whether readthrough or termination of transcription occurs by interacting with unacylated or acylated tRNA, respectively. Our goal is to uncover a new class of small molecules that will disrupt the binding of tRNA to the 5’UTR and through that, inhibit transcription. We are creating a transcription based reporter assay to examine small molecules for their ability to disrupt gene expression. We cloned the entire 5’UTR of a B. subtilis aaRS gene upstream of the adenine riboswitch aptamer, with an intervening linker region. This aptamer binds 2-aminopurine and upon binding, quenches the fluorescence normally associated with this molecule. We have successfully transcribed the chimeric RNA. Preliminary fluorescence data shows the aptamer RNA is able to quench 2- aminopurine (2AP) fluorescence. Future work includes carrying out the fluorescence studies in real time during transcription and testing a library of small molecules to assess their ability to inhibit tRNA-mediated transcription.
Modi, Nishtha, "Probing the T-Box Riboswitch: A Novel, High-Throughput Transcription Reporter Assay Using a Fluorophore-Binding Aptamer" (2012). Biological Sciences. 15.