In vitro telomerase activity of paired element disruption mutants. (A) Telomerase activity immunopurified from wild-type, DMA, DMB, and DMC mutants. Extracts were prepared from a strain expressing ProA-tagged Est2p and wild-type, DMA, DMB, or DMC mutant TLC1 RNA. Extracts were then incubated with IgG beads to immunopurify telomerase. Beads were assayed for in vitro telomerase activity by incubation with 2.5 μM telomeric primer, 100 μM TTP, and 1.7 μM [α-32P]dGTP, with 5 μL of wild-type beads, 2.5 μL of DMC, and 10 μL each of DMA or DMB beads assayed. Telomerase products were resolved on a 12% PAGE-7 M urea sequencing gel. (Loading ctrl) A control for product recovery: a 32P 5′-end-labeled 100-nt oligonucleotide added immediately after the reactions were stopped and visualized on the same gel. (+1 marker) in lane M was the 14-nt single-stranded telomeric primer extended by 1 nt with the addition of [33P]ddTTP and terminal deoxytransferase. (B) Coimmunoprecipitation of telomerase RNA mutants with ProA-Est2p. RNA coimmunoprecipitated with ProA-Est2p on IgG beads was isolated (as described for A), and analyzed by Northern blotting (as described for Fig. 2C). Blots were probed with radiolabeled TLC1 and U1 gene probes. (I) Input RNA; (FT) flowthrough from beads; (B) RNA bound to beads. The markers shown were radiolabeled [phis]X174-HaeIII-digested DNA fragments. (% loaded) Percentage of input, flowthrough, and bound RNAs loaded onto the gel. (C) Telomerase activity in the presence of all four nucleotides, and the effect of overexpression of TLC1 RNA and ProA-Est2p. Reactions were performed in the absence or presence of both 100 μM dATP and dCTP (lanes 1–5 and 6–10, respectively). In all cases, reactions contained 100 μM TTP and 0.9 μM [α-32P]dGTP. For the RNase A control reactions, beads were incubated with RNase A prior to the addition of the activity assay components.
