A mutant of Tetrahymena telomerase reverse transcriptase with increased processivity
A mutant of Tetrahymena telomerase reverse transcriptase with increased processivity
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DOI：
10.1074/jbc.m003246200
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发表时间：
2000-08-04
影响因子：
4.8
通讯作者：
Cech, TR
中科院分区：
文献类型：
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作者：
Bryan, TM;Goodrich, KJ;Cech, TR
端粒酶蛋白催化亚单位(TERT)是一种逆转录酶，它利用内部RNA分子作为模板来延长染色体DNA末端。在所有逆转录病毒RTS中，在催化天冬氨酸之前存在一个保守的酪氨酸，在TERT中，C基序是催化活性的关键基序，然而，这个位置是亮氨酸、缬氨酸或苯丙氨酸。我们建立并鉴定了一个强大的四膜虫端粒酶体外重组系统，并测试了氨基酸取代对活性的影响。在基序C中替换反转录病毒样酪氨酸并没有改变整体酶的活性，但增加了加工性，这种加工性的增加与对端粒DNA引物的亲和力增加有关。丙氨酸的取代不会增加加工性，而苯丙氨酸的取代有中间效应。这些数据表明，与其他RTS一样，这种氨基酸参与了端粒酶与引物的相互作用，并表明将一个氨基酸突变为逆转录病毒RTS中保守的氨基酸，使端粒酶与这些其他RTS更相似。
The protein catalytic subunit of telomerase (TERT) is a reverse transcriptase (RT) that utilizes an internal RNA molecule as a template for the extension of chromosomal DNA ends. In all retroviral RTs there is a conserved tyrosine two amino acids preceding the catalytic aspartic acids in motif C, a motif that is critical for catalysis, In TERTs, however, this position is a leucine, valine, or phenylalanine, We developed and characterized a robust in vitro reconstitution system for Tetrahymena telomerase and tested the effects of amino acid substitutions on activity. Substitution of the retroviral-like tyrosine in motif C did not change overall enzymatic activity but increased processivity, This increase in processivity correlated with an increased affinity for telomeric DNA primer. Substitution of an alanine did not increase processivity, while substitution of a phenylalanine had an intermediate effect. The data suggest that this amino acid is involved in interactions with the primer in telomerase as in other RTs, and show that mutating an amino acid to that conserved in retroviral RTs makes telomerase more closely resemble these other RTs.