FIG. 3.

Yeast telomerase containing a mutation at Est2p residue E76 does not exhibit a detectable decrease in product association in a primer competition assay. Extract was isolated from strain YKF120 (est2::HIS3) containing plasmids expressing either wild-type, protein A-tagged EST2 (ProA-EST2, lanes 1 to 10) or protein A-tagged est2^E76K (ProA-est2^E76K, lanes 11 to 13), and telomerase was immunopurified on IgG beads. Primer sequences are shown (top). Primers 14 and 29 contain the canonical telomeric sequence, while primer 5′NT contains nontelomeric sequence at its 5′ end (underlined). Telomerase assays were performed using the indicated combinations of primers (see boxes). The top primer was added at the beginning of the reaction, while the bottom primer (or buffer alone; empty box) was added after the reaction had proceeded for 7 min. Products resulting from extension of the 29-nucleotide primer are indicated with brackets. Lane 1 contains a sample that was pretreated with RNase A to eliminate telomerase activity.