Telomerase is an enzyme that maintains telomeres, the protective structures at the ends of chromosomes. Telomerase dysfunction is associated with premature aging disorders and inappropriate telomerase activation is associated with ~90% of all cancers, which has motivated studies to better understand the function of this crucial enzyme. Telomerase has two essential components, the protein component telomerase reverse transcriptase (TERT) and the telomerase RNA (TER). TERT functions by reverse transcribing a small template region of TER into telomere DNA. TER also has several conserved secondary and tertiary structures known to be important for telomerase function.
In this thesis research, I describe our attempts to better understand how conserved elements of TERT and TER assemble to form a functional enzyme. In Chapter II, using the well-characterized model system Tetrahymena thermophila, I describe our characterization of the assembly protein p65. p65 functions in Tetrahymena by binding TER and reorganizing important RNA motifs, placing them in the correct orientation to facilitate TERT binding. We identified a C-terminal domain of p65 which is necessary and sufficient for this activity. We then further characterized this domain, demonstrating that it binds a conserved region of the RNA and affects basepairing interactions in an important stem-loop region of the RNA. This work was originally published in the journal RNA.
In Chapter III, I further explain our efforts to characterize TERT-TER interactions. In this work we used a biochemical technique known as site-directed hydroxyl radical probing to identify sites of interaction between protein and RNA within the enzyme. Our work uncovered a specific interaction between a conserved RNA element known as the template boundary element (TBE) and a conserved protein element known as the CP2 motif. Mutagenesis experiments identified the most crucial amino acid determinants of the CP2 motif and demonstrated that CP2 motif mutations severely affect telomerase activity. This work originally appeared in the Journal of Biological Chemistry.
Another focus of my thesis work has been telomerase dynamics. Telomerase activity involves coordinated motions between protein, RNA, and DNA subunits within the holoenzyme. Understanding these dynamics is crucial to our understanding of telomerase function. In order to study dynamics within the telomerase holoenzyme, we used a technique known as single-molecule Förster resonance energy transfer (smFRET). FRET describes the distance-dependent energy transfer between a donor and acceptor fluorophore at sub-nanometer distances. When these fluorophores are site-specifically incorporated into a molecule of interest, real-time changes in FRET can be used to measure conformational changes within the molecule.
In Chapter IV, I describe a smFRET telomerase binding assay we developed to measure conformational motions during telomerase catalytic activity. We identified reciprocal motions in telomerase RNA on either side of the template that occur during telomerase extension activity. Using smFRET, we demonstrated that RNA on either side of the template undergoes a cycle of expansion and compactions as template RNA is pulled through the active site. This work was originally published in Nature Structural and Molecular Biology.
In Chapter V, I describe smFRET experiments which demonstrate that DNA bound to telomerase is an equilibrium between two conformations: an active state and an alternative state. Furthermore, we discovered that a conserved domain of TERT, the telomerase essential N-terminal (TEN) domain, is responsible for stabilizing the active state and TEN domain mutations which favor the alternative state disrupt the rate of telomerase processivity. Our work conclusively identifies a new DNA binding mode for telomerase and demonstrates an unappreciated function for a conserved telomerase protein domain.
In Chapter VI of this thesis, I describe preliminary work we have performed to further study transitions between the active and the alternative state described in Chapter V. I describe efforts to develop new methodologies to study FRET dynamics across the DNA primer, as well as our efforts to study protein-DNA and protein-RNA motions. These experiments are too preliminary to yield conclusive results, however they demonstrate great promise for future smFRET studies of telomerase.
In the final chapter of this thesis, I describe potential future experiments on telomerase. Having extensively discussed the TEN domain in Chapters V and VI, I focus on potential studies on real-time measurements of telomerase extension activity. I also discuss experiments on further structural characterization of protein-RNA interactions in the telomerase holoenzyme. I feel both of these topics merit further study in order to better understand the mechanism of this important enzyme.