Figure 2.

RNA sequence specificity of telomerase RNP reconstitution in lysate. Five microliters of lysate-expressed TERT was supplemented with 500 ng of the indicated competitor RNA and then with 10 ng of telomerase RNA. Samples were incubated at 30°C for 30 min then assayed for telomerase activity with the primer (TG)₈TTG. (Lane 1) No competitor; (lane 2) a telomerase RNA variant missing stem–loop IV, which binds TERT but is less active than the wild-type RNA (see Results); (lanes 3,4) tRNA; (lanes 5,6) 5S RNA; (lanes 7,8) total yeast RNA; (lanes 9,10) a 24-nucleotide RNA oligonucleotide representing telomerase RNA residues 36–59 which have no activity (Collins and Gandhi 1998). In some experiments, total yeast RNA stimulated telomerase activity to the same level as tRNA (not shown). Products corresponding to primer +1 and +6 nucleotides are indicated.