Poster Presentation 30th Lorne Cancer Conference 2018

Electron microscopy of human telomerase (#141)

Scott Cohen 1 , Rosalba Rothnagel 2 , Tram Phan 3 , George Lovrecz 3 , Tim Adams 3 , Tracy Bryan 1 , Michael Parker 4 , Ben Hankamer 2
  1. Children's Medical Research Institute, Westmead, NSW, Australia
  2. Institute for Molecular Bioscience, University of Queensland, St. Lucia, QLD, Australia
  3. CSIRO Manufacturing, Parkville, VIC, Australia
  4. St. Vincent's Institute for Medical Research, Fitzroy, VIC, Australia

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.

 

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; this system is providing sufficient telomerase for electron microscopy studies. We have determined a low-resolution structure by negative-stain EM, revealing an elongated, bilobal structure. We are currently in the early stages of imaging with cryo-EM, experimenting with functionalised derivatives of graphene to immobilise particles on the grid before vitrification. Our long-term aim is to apply structure-guided design to the development of small-molecule telomerase inhibitors.

 

(1) Cohen SB, et al. (2007) Science, 315, pp 1850-1853.