Flexibility of telomerase in binding the RNA template and DNA telomeric repeat.
Flexibility of telomerase in binding the RNA template and DNA telomeric repeat.
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DOI：
10.1073/pnas.2116159118
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发表时间：
2022-01-04
影响因子：
11.1
通讯作者：
Yang W
中科院分区：
文献类型：
--
作者：
Choi WS;Weng PJ;Yang W
端粒酶逆转录酶(TERT)在活性部位附近有一个保守的中心空腔。利用酶分析和突变分析，我们提供了实验证据，证明在DNA引物中人工循环的端粒重复序列可以瞬时地适应于卡氏赤霉菌(TcTERT)的腔内。从动力学上讲，tcTERT需要RNA模板和DNA引物之间至少4个核苷酸才能有效地合成DNA。在柔性连接物之后的RNA模板区域下游的RNA双链提高了通过tcTERT进行的引物延伸的效率。除了在每个端粒重复合成过程中容纳环出RNA的外围空腔外，能够容纳环出DNA的中央空腔可能有助于端粒重复合成周期之间的RNA模板转位。端粒酶通过重复反转录短RNA模板在线性染色体末端合成端粒。人端粒酶和四膜虫端粒酶的晶体结构(TcTERT)和低温电子显微镜(Cryo-EM)结构揭示了逆转录酶结构域的保守特征，包括在DNA 3‘端附近有一个空洞和与RNA模板的紧密相互作用。为了使RNA模板转移，它需要不配对并从DNA产物中分离出来。在这里，我们研究结构腔的潜力，以容纳一个环状的DNA凸起，并使RNA/DNA杂交体能够分离。以tcTERT为模型系统，我们证明了DNA引物中的环状端粒重复可以被tcTERT调节和延伸，但不能被逆转录病毒逆转录酶调节和延伸。减少空腔大小的突变降低了tcTERT延伸环状DNA底物的能力。与端粒酶的低温EM结构一致，我们发现tcTERT需要在RNA模板和DNA引物之间至少4个核苷酸才能有效地合成DNA。我们还测定了tcTERT的三元复合结构，其中包括2.0 ra分辨率的下游RNA/DNA杂交体，并表明下游RNA双链，相当于端粒酶RNA中的5‘模板-边界元件，提高了tcTERT合成端粒的效率。虽然TERT有一个预先形成的活性部位，没有开放和关闭的构象变化，但它含有空腔来容纳环出的RNA和DNA。灵活的RNA-DNA结合可能是端粒重复加成过程的基础。
Telomerase reverse transcriptase (TERT) has a conserved central cavity near the active site. Using enzymatic and mutagenesis analyses, we provide experimental evidence that an artificially looped-out telomeric repeat in the DNA primer can be transiently accommodated in the cavity of Tribolium castaneum (tcTERT). Kinetically, tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. An RNA duplex downstream of the RNA-template region after a flexible linker enhances the efficiency of primer extension by tcTERT. In addition to the peripheral cavities that accommodate looped-out RNA during each telomeric repeat synthesis, the central cavity that can accommodate the looped-out DNA may aid RNA-template translocation between cycles of telomeric repeat synthesis. Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of Tribolium castaneum telomerase reverse transcriptase (tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and Tetrahymena telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3′ end and snug interactions with the RNA template. For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by tcTERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. We also have determined the ternary-complex structure of tcTERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5′ template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by tcTERT. Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA–DNA binding likely underlies the processivity of telomeric repeat addition.
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影响因子：
64.5
作者：
Wang F;Yang W
通讯作者：
Yang W
端粒DNA合成过程中端粒酶RNA定位的RNA手风琴模型。
影响因子：
16.8
作者：
Berman, Andrea J.;Akiyama, Benjamin M.;Stone, Michael D.;Cech, Thomas R.
通讯作者：
Cech, Thomas R.
影响因子：
2.9
作者：
Hammond, PW;Cech, TR
通讯作者：
Cech, TR
影响因子：
5.7
作者：
Rouda, Susan;Skordalakes, Emmanuel
通讯作者：
Skordalakes, Emmanuel
影响因子：
16.8
作者：
Jansson LI;Akiyama BM;Ooms A;Lu C;Rubin SM;Stone MD
通讯作者：
Stone MD