Two independent regions of human telomerase reverse transcriptase are important for its oligomerization and telomerase activity.
Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, contains motifs conserved among reverse transcriptases. Several nucleic acid-dependent polymerases that share a "fingers, palm, and thumb substructure" were shown to oligomerize. Here we demonstrate that hTERT also has this ability using partially purified recombinant hTERTs and mammalian cells co-expressing differently tagged hTERTs. Human template RNA (hTR), by contrast, has no effect on the structural oligomerization of hTERTs. Therefore, hTERT has an intrinsic ability of oligomerization in the absence of hTR. We identified two separate regions as essential for the oligomerization. The regions, amino acids 301-538 (amino-terminal region) and amino acids 914-928 (carboxyl-terminal region), are outside the fingers and palm substructure covering motif T to D and interact with each other in vivo. A substituted mutant of hTERT, hTERT-D712A-V713I, which was reported as a dominant negative form of hTERT, bound to the wild-type hTERT and inhibited its telomerase activity transiently expressed in telomerase-negative finite normal human fibroblast. The truncated forms of hTERT containing the binding region to the wild-type hTERT partially inhibited the telomerase activity, probably by preventing the wild-type hTERT from forming an oligomer. Taken together, the oligomerization of hTERT is an important step for telomerase activity.
Arai
Kuniaki
K
Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, Takarama-machi 13-1, Kanazawa 920-0934, Japan.
Masutomi
Kenkichi
K
Khurts
Shilagardy
S
Kaneko
Shuichi
S
Kobayashi
Kenichi
K
Murakami
Seishi
S
D020816:Amino Acid Motifs; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D015153:Blotting, Western; D019556:COS Cells; D002455:Cell Division; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D019098:Telomerase / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012313:RNA; D005982:Glutathione Transferase; D019098:Telomerase