TABLE 1.

Telomerase activity, hTERT/hTR association, and functional multimerization of hTERT N terminal mutants

MutationIdentity and conservation of deleted residuesa, bRegion of N terminuscTelomerase activitydhTERT/hTR associationeRIDfPhysical multimerization with GST-hTERT D868NfFunctional complementation of GST-hTERT D868Ng
AvgSDnAvgSDn
Wild type1.00051.0004++
D868N0041.050.123+
Δ30-39 RLGPQGWRLVN0.100.0240.810.103RID1++/−
Δ70-79 SFRQVSCLKEGQ motif0.380.1850.870.133RID1++
Δ110-119GPPEAFTTSVGQ motif (DAT domain)0.990.0630.830.113RID1++
Δ150-159VHLLARCALFGQ motif0040.910.023RID1++
Δ190-199ASGPRRRLGCGQ linker0.920.1651.110.193++
Δ230-239RSLPLPKRPRLinker0.870.2540.900.253++
Δ270-279FCVVSPARPALinker1.160.1851.010.263++
Δ310-319TSRPPRPWDTLinker0.700.4141.070.072++
Δ350-359LRPSLTGARRLinker (VSR motif)0.010.0130.550.082RID2+
Δ390-399 RPLFLELLGNLinker/CP motif0.040.0540.520.113RID2+
Δ430-439KPQGSVAAPE1.140.1840.950.033++
Δ481-490 R HNE R RFLRNQFP motif0.040.0430.410.14RID2+
Δ508-517 LT WKMSVRDCQFP motif0.150.1830.270.074RID2+
W547AT motif0.010.0130.430.104RID2+
W547FT motif1.000.0230.770.134RID2++
  • a Bold face indicates 100% conservation among vertebrate TERTs (Homo sapiens, Mesocricetus auratus, Mus musculus, and Xenopus) (17, 18, 20, 23, 26, 32, 33, 37). Underlined bold face indicates ≥70% conservation among all TERTs (H. sapiens, M. auratus, M. musculus, Xenopus, Arabidopsis, Schizosaccharomyces pombe, Euplotes, Oxytricha, Tetrahymena, S. cerevisiae, and Candida albicans) (46). Underlined lightface indicates >70% conservation of a residue among all TERTs but indicates <100% conservation among vertebrate TERTs.

  • b Alignments were performed with NCBI BLAST and by using the multiple sequence alignment published by Xia et al. (46). See Fig. 1 for sequence alignments of the extreme N-terminal (N) and linker regions of the vertebrate TERTs.

  • c Motifs identified by multiple sequence alignment (46). Additional domains/motifs identified in hTERT are indicated in parentheses.

  • d Telomerase activities of mutant telomerases reconstituted in RRL were expressed relative to the activity of wild-type enzyme.

  • e hTR association with hTERT mutants generated in RRL was expressed relative to hTR association with wild-type hTERT.

  • f The physical association of hTERT mutants with GST-hTERT D868N in vitro was evaluated by SDS-PAGE using (i) immunoprecipitated mixtures of yeast- or RRL-synthesized GST-hTERT D868N and RRL-synthesized hTERT mutants (Fig. 4C and 5A, respectively) and (ii) immunoprecipitated RRL cosynthesis reactions (Fig. 6).

  • g The ability of hTERT mutants to functionally complement inactive GST-hTERT D868N cosynthesized in RRL was evaluated by TRAP assay. The symbol “+” indicates functional complementation to reconstitute telomerase activity. The symbol “−” indicates an inactive mutant that does not complement GST-hTERT D868N. “+/−” indicates weak complementation.