The C terminus of the human telomerase reverse transcriptase is a determinant of enzyme processivity
The C terminus of the human telomerase reverse transcriptase is a determinant of enzyme processivity
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DOI：
10.1093/nar/gkg437
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发表时间：
2003-07-15
影响因子：
14.9
通讯作者：
Autexier, C
中科院分区：
文献类型：
--
作者：
Huard, S;Moriarty, TJ;Autexier, C
端粒酶 (TERT) 的催化亚基包含保守的逆转录酶样基序，但 N 端和 C 端区域是端粒酶独有的。尽管序列保守性较弱，但来自各种生物体的 TERT C 末端与端粒酶特异性功能有关，包括端粒酶活性、与其他 TERT 分子的功能多聚化、酶的持续合成能力和端粒长度维持。我们研究了含有小 C 端缺失或取代的 hTERT 蛋白，以鉴定和表征介导端粒酶活性、hTERT 多聚化和持续合成能力的 hTERT 结构域。通过对五种脊椎动物 TERT 和拟南芥 TERT 的序列比对，我们鉴定了人类端粒酶活性和功能性 hTERT 互补所需的高度保守氨基酸块。我们采用非基于 PCR 的端粒酶延伸测定来表征体外转录/翻译兔网织红细胞裂解物系统中表达和重构的端粒酶。使用该测定，我们发现 hTERT C 末端，如酿酒酵母 TERT 的 C 末端，有助于在单个 6 碱基端粒重复序列中连续添加核苷酸（I 型持续性）。 hTERT C 末端的某些突变也减少了多个端粒重复序列的重复添加（II 型持续性）。我们的结果表明 TERT C 末端在端粒酶持续合成能力中具有功能保守的作用。
The catalytic subunit of telomerase (TERT) contains conserved reverse transcriptase-like motifs but N- and C-terminal regions unique to telomerases. Despite weak sequence conservation, the C terminus of TERTs from various organisms has been implicated in telomerase-specific functions, including telomerase activity, functional multimerization with other TERT molecules, enzyme processivity and telomere length maintenance. We studied hTERT proteins containing small C-terminal deletions or substitutions to identify and characterize hTERT domains mediating telomerase activity, hTERT multimerization and processivity. Using sequence alignment of five vertebrate TERTs and Arabidopsis thaliana TERT, we identified blocks of highly conserved amino acids that were required for human telomerase activity and functional hTERT complementation. We adapted the non-PCR-based telomerase elongation assay to characterize telomerase expressed and reconstituted in the in vitro transcription/translation rabbit reticulocyte lysate system. Using this assay, we found that the hTERT C terminus, like the C terminus of Saccharomyces cerevisiae TERT, contributes to successive nucleotide addition within a single 6-base telomeric repeat (type I processivity). Certain mutations in the hTERT C terminus also reduced the repetitive addition of multiple telomeric repeats (type II processivity). Our results suggest a functionally conserved role for the TERT C terminus in telomerase enzyme processivity.