Mechanism of eubacterial 6S RNA release and Development of RNA Mango toolkit to study Ribonucleoprotein complexes

Ribonucleic acids (RNA) perform diverse biochemical functions in cells ranging from being an intermediate in the flow of genetic information, catalyst performing crucial reactions and as regulators of several processes. In bacteria, 6S RNA is a global transcription regulator that binds and inhibits house keeping RNA polymerase holoenzyme (core polymerase + σ70) under low nutrient conditions and rescues transcription in high nutrient conditions by the synthesis of a short product RNA (pRNA) using itself as a template. I show that a kinetic intermediate containing 6S RNA:core enzyme complex, that is enhanced by the formation of a phylogenetically conserved ‘release’ hairpin arises during 6S RNA release. Using nucleotide feeding experiments to slow down the release and a 6S RNA mutant which precludes the hairpin formation, I found the release process involves intrinsic ‘scrunching’ type mechanism that is modulated by the ability to form a release hairpin during the process of 6S RNA release. Given the importance of RNA in regulating various cellular processes, a fluorescence tool to track RNA in real time is limiting as RNA lacks intrinsic fluorescence. The Unrau lab has in vitro selected RNA Mango aptamer that binds thiazole orange with nanomolar affinity while increasing its fluorescence up to 1100 fold. To elucidate how this small aptamer exhibits such properties, which make it particularly well suited for studying low-copy cellular RNAs, we, in collaboration with D′Amaré’s lab, determined its co-crystal structure, discovering a three-tiered G-quadruplex connected to a duplex through a GAAA tetraloop-like junction. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb) ligand I have created a Mango toolkit that simultaneously enables the purification and characterization of endogenous cellular RNPs in vitro.