Telomeres, the repetitive DNA-protein complexes at the ends of linear chromosomes, shorten with each cycle of DNA replication, providing a counting mechanism to limit the number of times a cell can divide. Most cancer cells have activated the ribonucleoprotein enzyme telomerase to add telomeric DNA repeats and counteract telomere shortening, allowing for unlimited proliferation. Although inhibition of telomerase has been considered a promising approach to cancer therapy for more than two decades, its low cellular abundance (~50-100 copies/cell) and challenging biochemistry have stymied development of small-molecule inhibitors. Our long-term aim is to apply structure-guided design to the development of small-molecule telomerase inhibitors.
We reported the purification and composition of the core human telomerase enzyme complex, consisting of two molecules each of: i) the telomerase reverse transcriptase catalytic protein; ii) telomerase RNA; and iii) the RNA-binding protein dyskerin (1). Building on this knowledge we developed an over-expression system in suspension HEK-293T cells that yields ~400-fold greater activity over endogenous levels, providing sufficient telomerase for electron microscopy. We determined a low-resolution structure by negative-stain EM, revealing an elongated, bilobal structure that displays significant conformational flexibility along the central hinge between the two catalytic cores. We discovered a means to dissociate the enzyme into two active monomers, thereby circumventing the conformational flexibility of the dimer and greatly improving single-particle analysis.
We are currently in the early stages of imaging by cryo-EM. Having been stymied by aggregation and/or low particle density during conventional vitrification of thin aqueous films, we are making progress with immobilization of telomerase onto TEM-grids coated with single-layer transparent graphene-oxide.
(1) Cohen SB, et al. (2007) Science, 315, pp 1850-1853.