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首页> 外文期刊>Molecular and Cellular Biology >Functional Regions of Human Telomerase Reverse Transcriptase and Human Telomerase RNA Required for Telomerase Activity and RNA-Protein Interactions
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Functional Regions of Human Telomerase Reverse Transcriptase and Human Telomerase RNA Required for Telomerase Activity and RNA-Protein Interactions

机译：人端粒酶逆转录酶和端粒酶活性和RNA-蛋白质相互作用所需的人端粒酶RNA的功能区。

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摘要

Telomerase is a specialized reverse transcriptase (RT) that is minimally composed of a protein catalytic subunit and an RNA component. The RNA subunit contains a short template sequence that directs the synthesis of DNA repeats at the ends of chromosomes. Human telomerase activity can be reconstituted in vitro by the expression of the human telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. We analyzed telomerase activity and binding of hTR to hTERT in RRL by expressing different hTERT and hTR variants. hTRs containing nucleotide substitutions that are predicted to disrupt base pairing in the P3 helix of the pseudoknot weakly reconstituted human telomerase activity yet retained their ability to bind hTERT. Our results also identified two distinct regions of hTR that can independently bind hTERT in vitro. Furthermore, sequences or structures between nucleotides 208 and 330 of hTR (which include the conserved CR4-CR5 domain) were found to be important for hTERT-hTR interactions and for telomerase activity reconstitution. Human TERT carboxy-terminal amino acid deletions extending to motif E or the deletion of the first 280 amino acids abolished human telomerase activity without affecting the ability of hTERT to associate with hTR, suggesting that the RT and RNA binding functions of hTERT are separable. These results indicate that the reconstitution of human telomerase activity in vitro requires regions of hTERT that (i) are distinct from the conserved RT motifs and (ii) bind nucleotides distal to the hTR template sequence.
机译：端粒酶是一种专门的逆转录酶（RT），它最少由蛋白质催化亚基和RNA成分组成。 RNA亚基包含一个短模板序列，可指导染色体末端DNA重复序列的合成。可以在兔网织红细胞裂解液（RRL）系统中在重组人端粒酶RNA（hTR）存在下表达人端粒酶蛋白催化亚基（hTERT），在体外重建人端粒酶活性。我们通过表达不同的hTERT和hTR变体分析了端粒酶活性和rTR中hTR与hTERT的结合。 hTRs包含核苷酸取代，预计会破坏假结的P3螺旋中的碱基配对，弱重组人类端粒酶活性，但仍保留了其结合hTERT的能力。我们的结果还确定了hTR的两个不同区域，它们可以在体外独立结合hTERT。此外，发现hTR的核苷酸208和330之间的序列或结构（包括保守的CR4-CR5结构域）对于hTERT-hTR相互作用和端粒酶活性重构很重要。人TERT羧基末端氨基酸的缺失延伸至基序E或前280个氨基酸的缺失消除了人类端粒酶的活性，而不会影响hTERT与hTR缔合的能力，这表明hTERT的RT和RNA结合功能是可分离的。这些结果表明，在体外重建人端粒酶活性需要hTERT区域，即（i）与保守的RT基序不同，以及（ii）结合hTR模板序列远端的核苷酸。

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