Investigating The Function Of Conformational Heterogeneity In Telomerase Rna Using Multi-Dimensional Chemical Mapping And Single Molecule Spectroscopy

Cells preserve genomic integrity and prevent the DNA damage response at the ends of chromosomes by maintaining specialized chromatin structures called telomeres. Telomere DNA is synthesized by the reverse transcriptase telomerase, a multi-subunit ribonucleoprotein (RNP) enzyme. Telomerase RNA contains several structurally conserved domains that contribute to RNP biogenesis and function. One of these domains is the conserved region 4 and 5 (CR4/5), which folds into a three-way junction (3WJ), binds directly to the telomerase catalytic protein subunit, and is absolutely required for telomerase function in vitro and in vivo. Generally, 3WJs have been shown to be highly dynamic and capable of adopting multiple tertiary conformations. In the case of the CR4/5 domain, two dramatically different structures have recently been reported for the 3WJ in the absence or presence of its cognate protein. These structures highlight the ability of a particular RNA sequence to fold into exceedingly distinct structures. Precisely how the intrinsic folding properties of the CR4/5 domain contribute to functional RNP assembly remains to be elucidated. In the present work, we are characterizing the 3WJ RNA folds from several model telomerase systems using high-throughput chemical probing and the recently established mutate-and-map strategy. In this way, we will investigate the folding heterogeneity of telomerase 3WJs and will generate experimentally validated structural models for distinct RNA conformations present in the equilibrium ensemble. To complement these bulk biochemical experiments, we use single-molecule FRET, a technique that is uniquely suited for studying dynamic and heterogeneous RNAs. Taken together, these studies will shed light on mechanisms of telomerase RNA folding and RNP assembly, as well as more fundamental principles of RNA structural dynamics and function.