| Telomerase is a ribonucleoprotein (RNP) that, together with telomeres, protects the ends of linear chromosomes from replicative erosion. Though crucial for survival in most eukaryotes and implicated in both aging and cancer, little is known about the structural details of the enzyme. In this thesis, I present three fluorescence-based, single-molecule studies that determined a hierarchical telomerase assembly pathway, resolved conformational details of a telomerase RNA motif, and revealed global structural rearrangements instigated by RNA--protein interactions.;Following an overview of telomerase biology and single-molecule, fluorescence resonance energy transfer (smFRET) methods in Chapter 1, I establish in Chapter 2 the hierarchical assembly of the telomerase holoenzyme. A folding cofactor, p65, binds to a conserved helical domain of telomerase RNA and induces a conformational rearrangement that brings the distal portion of the stem (Stem IV) towards the active site. A conserved bulge in Stem IV is required for this rearrangement. With Stem IV thus repositioned, the catalytic subunit telomerase reverse transcriptase (TERT)---readily assembles with telomerase RNA, further compacting it.;In Chapter 3, I discuss a structural and functional analysis of a catalytically important motif, the telomerase RNA pseudoknot. The region is stably folded when free in solution, but is misfolded in full-length, protein-free RNA. Assembly with TERT is necessary to rescue the pseudoknot structure from competing interactions with other parts of the RNA. A single-molecule structure-function assay demonstrates stably folded pseudoknot is obligatory for telomerase activity.;Mutations in Loop IV of Stem IV have been proposed to perturb folding of the pseudoknot region, and their impact on telomerase conformation and activity is reported in Chapter 4. While no alteration of Loop IV disrupts pseudoknot structure, certain Loop IV mutations can completely abolish Loop IV---TERT interaction that normally positions Loop IV close to the active site. The severity of this structural defect is proportional to perturbations in activity and can be mitigated by p65 in the case of single-nucleotide substitutions. Complete substitution of Loop IV, however, cannot be rescued.;In Conclusions, I integrate the above studies and present a global model of the telomerase RNP conformation and assembly. |
