Fig. 4. Telomerase forms a dimer. (A) Scheme for the method used to determine the number of RNA molecules in the telomerase RNP (see text). (B) Affinity purification allows sequence-specific selection of telomerase complexes containing different template sequences. Telomerase was reconstituted with either wild-type or mutant hTER (template sequence: 5′-GAUUGGGAUU-3′), respectively. One half of the mixture was subjected to a purification specific for wild-type hTER and the other half to a purification specific for mutant hTER. The amount of hTER RNA was measured using quantitative RT–PCR following immunoprecipitation with αhTERT antibodies (+) or rabbit IgG (–). (C) Telomerase contains more than one hTER subunit. The experiment depicted schematically in Figure 3A was performed with different mixtures of wild-type and mutant RNA (lanes 3–8, lanes 11–16). RT–PCR products derived from wild-type hTER were cleavable with DdeI (lane 1 and 2) whereas RT–PCR products derived from mutant hTER were lacking this site (lanes 9 and 10). The band that has a slightly lower mobility than the uncleaved/mutant PCR products corresponds to heteroduplex DNA consisting of one wild-type and one mutant strand. (D) Quantitation of the experiment shown in Figure 4C. Data points (open diamonds) with standard deviations and a best-fitted solid line are shown together with theoretical curves (dotted lines) for telomerase being a monomer (labeled with one filled circle), dimer (two filled circles), trimer (three filled circles) or tetramer (four filled circles).