The T box transcription antitermination riboswitch regulatesgene expression by the interaction of uncharged cognate tRNA with the 5� untranslated region of the mRNA during transcription. This interactioninvolves, in part, the base pairing of the tRNA accepter end with four bases inthe bulge region of the T box antiterminator RNA element. This interactionprevents the formation of an alternative terminator element and results incomplete transcription of the gene. We developed a fluorescence anisotropyassay using fluorescently labeled RNA model AM1A to monitor tRNA binding to theT box anititerminator. The choice of fluorophor was optimized and the assay wasused to identify ligands that disrupt the tRNA-T Box antiterminator RNAinteraction. Two sets of small molecules were screened in a multi-well platearray and several compounds were identified with greater than 60% inhibitionefficiencies. The interaction between the lead compounds and T Boxantiterminator model RNA was studied in more detail using FluorescenceResonance Energy Transfer (FRET) and enzymatic probing.

Fluorescence anisotropy screening for inhibitors of tRNA-T
Box antiterminator RNA riboswitch interaction

239th National Meeting (2010)

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Fluorescence anisotropy screening for inhibitors of tRNA-T Box antiterminator RNA riboswitch interaction