Tetrahymena strain constructions and steps of tag-based affinity purification were done as described6 (link) and in Methods. To label the tagged subunit for EM, telomerase particles were first purified using anti-FLAG M2 antibody resin then bound to rabbit-IgG resin. The telomerase-bound IgG resin was then incubated with Fab derived from anti-FLAG M2 IgG, and elution was effected by protease cleavage. Negatively stained EM specimens were prepared with fresh telomerase samples, stained with 0.8% uranyl formate, and examined with an FEI Tecnai F20 electron microscope operated at 200 kV. Frozen hydrated specimens were prepared using Quantifoil grids and imaged with an FEI Titan Krios electron microscope operated at 120 kV. The image processing tasks, including image classification and RCT reconstruction, were performed as described in Methods.
Telomerase activity assays were performed at room temperature using purified telomerase complexes on FLAG antibody resin with standard Tetrahymena holoenzyme reaction conditions using 0.3 μM 32 (link)P-labeled dGTP. Holoenzyme reconstitution used synthetic genes encoding TERT-f, p75, p65, p50, p45, and p19 for expression in RRL; TER purified following in vitro transcription by T7 RNA polymerase; and N-terminally His6-tagged Teb1BC purified following bacterial expression21 (link).
Full Methods and any associated references are available in the online version of the paper.
Telomerase activity assays were performed at room temperature using purified telomerase complexes on FLAG antibody resin with standard Tetrahymena holoenzyme reaction conditions using 0.3 μM 32 (link)P-labeled dGTP. Holoenzyme reconstitution used synthetic genes encoding TERT-f, p75, p65, p50, p45, and p19 for expression in RRL; TER purified following in vitro transcription by T7 RNA polymerase; and N-terminally His6-tagged Teb1BC purified following bacterial expression21 (link).
