FIG. 3.

Role of the P3 helix of the hTR pseudoknot in telomerase function and two independent hTERT binding sites within hTR. (A) Northern blot of total RNA harvested from RRLs in which hTERT was synthesized in the absence of hTR (lane 1) or presence of wild-type hTR (lane 2) and hTR variants. The Northern blot was probed for hTR-specific sequences. M, DNA markers (sizes, in base pairs, are on the left). RNAs were separated by electrophoresis on a 6% acrylamide–7 M urea gel. (B and C) Equal volumes of RRL reaction products generated in the absence (lanes 11 and 22) or presence of the different hTR variants were subjected to immunoprecipitation (IP) with an antibody to the T7 tag. The washed beads were analyzed for telomerase activity (B) and hTERT-hTR coimmunoprecipitation (C). For panel B, telomerase activity was analyzed by the TRAP assay, and 100 ng of partially purified 293 cell extracts were used as a positive control (lane 21). IC, internal PCR control; WT, wild-type. For panel C, hTERT-hTR coimmunoprecipitation was analyzed by Northern blotting using an hTR-specific probe. The arrowheads indicate the positions at which full-length hTR (FLhTR), hTR164-330, and hTR33-147 migrate.