UC Santa Cruz

Telomerase is a unique eukaryotic enzyme responsible for maintaining the genomic integrity of chromosome ends, through the addition of protective DNA sequences called telomeres. The telomerase holoenzyme is composed of a protein component called TERT, and an RNA component called TR. TERT is a well conserved protein that contains many domains, one of which is the reverse transcriptase responsible for processive addition of telomere repeats to the chromosomal DNA. TER carries several necessary functional regions, which play critical roles in the activity of telomerase. These include the RNA template that is used as the basis for telomere replication, as well as a pseudoknot fold and a three-way junction called CR4/5. Overall TER displays much less sequence conservation than its protein counterpart, but its functional domains bear significant structural similarity throughout Eukarya. The CR4/5 region plays a significant, yet enigmatic role in the mechanism of telomerase. In this work I aim to further define the functional roles of the CR4/5 region through study of its structural dynamics, using single-molecule FRET. I investigate the conformational interconversions displayed by the yeast Kluyveromyces lactis CR4/5, and the impact that magnesium and junction sequence have on these dynamics. Further, I examine how models of the K. lactis CR4/5 correspond to the solved structures of CR4/5 from other organisms, how the dynamics found in K. lactis can be related to these organisms, and what insights are gained by real-time observations of this region. Lastly, I discuss the potential roles played by the CR4/5 region in the overall telomerase mechanism