Telomerase RNA Bound by Protein Motifs Specific to Telomerase Reverse Transcriptase  Tracy M. Bryan, Karen J. Goodrich, Thomas R. Cech  Molecular Cell  Volume 6, Issue 2, Pages 493-499 (August 2000) DOI: 10.1016/S1097-2765(00)00048-4

Figure 1 Summary of Tetrahymena TERT Mutations Analyzed in This Study and Their Telomerase Activity and Extent of Binding to the Telomerase RNA (A) Sequences of the conserved motifs in the Tt_TERT protein. Distances (in amino acids) between motifs and to the ends of the protein are shown. A consensus derived from nine TERT sequences is shown above (the nine proteins being those from the ciliated protozoa T. thermophila, Oxytricha trifallax, and E. aediculatus, the yeasts S. cerevisiae, Candida albicans, and S. pombe, the mammals Homo sapiens and Mus musculus, and the plant Arabidopsis thaliana). Amino acids are included in the consensus if they appear in at least 7 of the 9 sequences and are typed in bold if the remaining amino acids are conservative substitutions. Colored residues are conserved throughout all nine sequences. Asterisks indicate amino acid similarity in all nine sequences (as defined by the program Clustal, using a Blosum matrix). A consensus derived from just the three ciliated protozoa sequences is also shown for motif CP. Boxed amino acids are those mutated to alanine in this study. The arrows indicate the three aspartates necessary for catalysis. Numbered lines beneath the sequence represent block mutations. (B) Summary of telomerase activity and RNA binding ability of one double and 27 single amino acid substitution mutants. Colored amino acids are those conserved in all nine TERT sequences. Telomerase activity and RNA binding were measured using the assays shown in Figure 2 and Figure 3, respectively. Each mutant was quantitated by comparison to wt TERT on the same gel. Each experiment was carried out at least twice, and the mean of the experiments was scored according to the following scale: (−) < 1% of wild type; (+) 1% –10% of wild type; (++) 10%–50% of wild type; (+++) 50%–80% of wild type; (wt) >80% of wild type. Telomerase activity is shown for immunopurified enzyme only. aTelomerase activity of these immunopurified enzymes was significantly less than that in reticulocyte lysates (p < 0.02 in a t test). bTelomerase activity of these immunopurified enzymes was significantly greater than that in reticulocyte lysates (p < 0.05 in a t test). (C) Telomerase activity and RNA binding ability of 10 multiple amino acid mutants. Mutants 2 and 3 have alanine substituted for 4 and 10 amino acids in the CP motif, respectively. In mutant 4, the sequence of the 10 underlined amino acids in the CP motif is reversed. The other 7 mutants have deletions of the regions indicated in (A). Telomerase activity and RNA binding were quantitated relative to wt and the results scored according to the scale in (B). Molecular Cell 2000 6, 493-499DOI: (10.1016/S1097-2765(00)00048-4)

Figure 2 Telomerase Activity of Selected TERT Mutants The indicated mutants were expressed in reticulocyte lysates, immunopurified, and assayed for the ability to extend a (GGGGTT)3 DNA primer. LC, an end-labeled 100-mer oligonucleotide used as a recovery and loading control. +1, position of DNA primer with one added nucleotide. Molecular Cell 2000 6, 493-499DOI: (10.1016/S1097-2765(00)00048-4)

Figure 3 Assay for Ability of TERT Protein to Immunoprecipitate Telomerase RNA The indicated mutants were expressed in reticulocyte lysates in the presence of [35S]methionine and 32P-end-labeled telomerase RNA. After immunoprecipitation with an antibody to the T7 tag on the TERT protein, samples were run on an SDS-PAGE gel that was dried and exposed to a phosphorimager screen. The locations of the 35S-labeled TERT protein and 32P-labeled RNA are indicated (upper panel). The lower panel shows an exposure of the same gel with a piece of X-ray film between the gel and the phosphorimager screen in order to screen out signal from the 35S-labeled protein. –prot, a control with telomerase RNA but no TERT protein, to measure nonspecific binding of RNA to the immunoprecipitation beads. Molecular Cell 2000 6, 493-499DOI: (10.1016/S1097-2765(00)00048-4)

Figure 4 Partial Proteolysis of WT and Mutant TERT with LysC TERT was expressed in reticulocyte lysates in the presence of [35S]methionine, immunopurified, and incubated with LysC for the times indicated. Proteolysis products were separated by SDS-PAGE before exposure to a phosphorimager screen. ΔCP and ΔT contain deletions of the underlined amino acids labeled 1 and 5, respectively, in Figure 1A. Molecular Cell 2000 6, 493-499DOI: (10.1016/S1097-2765(00)00048-4)