Date of Award

Spring 1-1-2012

Document Type


Degree Name

Doctor of Philosophy (PhD)

First Advisor

Deborah S. Wuttke

Second Advisor

Mark Winey

Third Advisor

Shelley D. Copley

Fourth Advisor

Andreas Hoenger

Fifth Advisor

Thomas Blumenthal


Telomeres are the nucleoprotein complexes at the ends of linear chromosomes that protect the genome from degradation and chromosomal fusions. Telomeres are replicated by the specialized enzyme telomerase. The telomerase holoenzyme in S. cerevisiae contains an RNA template and three known protein subunits, Est1, Est2 and Est3 (est = ever shorter telomeres for the phenotype observed upon their deletion). The reverse transcriptase Est2 and the RNA template TLC1 constitute the catalytic core of the telomerase holoenzyme. While Est1 and Est3 are not required for catalysis in vitro, they are strictly required for activity in vivo. The function of Est3 has remained elusive, although genetic data suggests that one mode of Est3's function might be carried out via its interaction with Est2.

To provide insights into Est3 function, we have solved its high resolution structure. Because Est3 is a difficult protein target for structure elucidation, the structure was solved using a relatively novel strategy of combining minimal NMR experimental data (chemical shifts, RDCs and NOEs) with Rosetta de novo structure prediction. The structure is an OB-fold, with five-stranded β-barrel, capped with an α-helix and has some specialized features that distinguish it from other OB folds. The canonical loop L45 is quite unusual in the case of Est3, in that it is long and highly structured and plugs on top of the OB-fold canonical ligand binding face. Even in the absence of appreciable sequence relationship, the Est3 structure shows remarkable similarity to HsTPP1-OB structure, not only in the core β-barrel, but also in the positioning of the α-helix at the base and placement of C-terminal tail partially covering the canonical OB-fold binding face. Mapping residues involved in telomerase-association onto the structure reveals a novel protein interaction surface at the base of the β-barrel for Est3 and TPP1-OB. In vivo analysis, using structure-guided mutagenesis of Est3 surface also identified several new, functionally relevant, residues of Est3. The structure also served as a validation tool for an in vivo guided in vitro study that showed a direct correlation of in vivo dominant-negative mutants with their structural retention in vitro.


Sixth advisor: Arthur Pardi.