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 thereby counteract telomere shortening, allowing for unlimited proliferation. In contrast, normal cells have undetectable or low levels of telomerase. Inhibition of telomerase is therefore a promising avenue for future anticancer therapy that should be effective against a broad range of cancers while displaying few side effects.
In 2007 we reported the purification and composition of the human telomerase enzyme complex, consisting of two molecules each of: i) the telomerase reverse transcriptase catalytic protein component; ii) the telomerase RNA; and iii) the RNA-binding protein dyskerin (1). Building on this knowledge we established an over-expression system in suspension HEK-293T cells that yields ~500-fold greater activity over endogenous levels, performed on the 20-Litre scale; this system has provided sufficient telomerase for negative-stain electron microscopy. We collected uranyl formate-stained images of purified telomerase and have processed ~25,000 particles to provide a low-resolution (~30 Angstrom) structure, revealing an elongated bi-lobal structure that displays significant conformational heterogeneity. Current efforts aim to obtain cryo-EM data to enable a higher-resolution structure.
(1) Cohen SB, et al. (2007) Science, 315, pp 1850-1853.