Abstract
Telomerase is an enzyme responsible for the maintenance of eukaryotic chromosome ends. The two main components required for activity are a protein subunit, the human telomerase reverse transcriptase (hTERT), and an RNA subunit, the human telomerase RNA (hTR). While telomerase is not active in most normal human cells, roughly 85% to 90% of oncogenic cells display increased telomerase activity. An understanding of the biochemistry of telomerase will aid in the development of molecules that will lead to antitumor-specific therapies. The first part of the work presented here describes a biochemical analysis of an RNA-RNA interaction between two catalytically important domains of hTR. The interactions were characterized via mobility-shift assay, mutation analysis, and UV cross-linking experiments. The data argue for a revised model for the structure of hTR, and point to possible three-dimensional contacts present in the telomerase complex. The next part of this thesis describes an in vitro selection against the catalytically important RNA stem-loop P6.1 in hTR. The in vitro selection was performed using mRNA display, which allows us to isolate RNA-binding peptides from libraries containing trillions of unique sequences. Unexpectedly, the selected peptide binds with high affinity and specificity to the relatively rare dimeric form of the P6.1 stem-loop rather than the more abundant monomeric conformation. Characterization of this novel RNA-peptide interaction was performed via circular dichroism, steady-state fluorescence, mobility-shift assay, and surface plasmon resonance. The data highlight the power of mRNA display to isolate high affinity ligands from large libraries of molecules.
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