Figure 6. Telomerase RNP assembly and activity without the TEN domain linker.
(A) Schematic representation and expression of N-terminally F-tagged human TERT proteins with the linker replaced by 20, 10, or 5 repeats of the sequence NAAIRS (TERT-20N through 5N) or linker deleted without compensating sequence insertion (TERT-∆PAL). TERTs expressed in 293T cells were detected by immunoblot with TERT antibody, and TERTs expressed in RRL were detected by 35S-methionine labeling during synthesis. (B) Activity and hTR content of 293T- or RRL-reconstituted, F-purified TERT RNPs with altered linker sequence, bound to FLAG antibody resin. Spot-blot hybridization was used to detect hTR. Relative activity and hTR content were normalized to the WT TERT purification after background subtraction of activity or hTR in the purification of untagged WT TERT. Specific activity was calculated from relative activity and relative hTR. (C) Processive extension of 5′-labeled (T2AG3)3 primer by telomerase assembled with WT, ∆PAL, or 20N TERT bound to FLAG antibody resin. The labeled primer was extended for 5 min before chase addition of unlabeled primer for a total extension time of 10, 20, or 40 min. (D) Activity and hTR content of telomerase in 293T input extracts or bound to Myc antibody resin. TPP1 OB-fold domain expression and purification were confirmed by immunoblot detection of the 3xMyc tag. Immunoblot and activity assay with whole-cell extract used 2% of the total purification input. Half of the post-purification sample was used for activity assays and half for Myc immunoblot. Spot-blot hybridization was used to detect hTR. Relative activity and hTR content were normalized to the input or bound sample for TPP1 purification of WT TERT, after bound hTR background subtraction using the purification without tagged TPP1 OB-fold domain. Relative percentage enrichment was calculated as relative bound activity adjusted for relative input activity. Specific activity was calculated from relative activity and relative hTR. (E) SDS-PAGE analysis of O-purified 293T MCP-TERT complexes labeled using CoA-Cy5. MCP-TERT fragments resulting from proteolysis within the PAL of WT TERT are schematized, in comparison to full-length TERT.