Telomeres safeguard the genome by suppressing illicit DNA damage responses at chromosome termini. In order to compensate for incomplete DNA replication at telomeres, most continually dividing cells, including many cancers, express the telomerase ribonucleoprotein (RNP) complex. Telomerase maintains telomere length by catalyzing de novo synthesis of short DNA repeats using an internal telomerase RNA (TR) template. TRs from diverse species harbor structurally conserved domains that contribute to RNP biogenesis and function. In vertebrate TRs, the conserved regions 4 and 5 (CR4/5) fold into a three-way junction (TWJ) that binds directly to the telomerase catalytic protein subunit and is required for telomerase function. We have analyzed the structural properties of the human TR (hTR) CR4/5 domain using a combination of in vitro chemical mapping, secondary structural modeling, and single-molecule structural analysis. Our data suggest the essential P6.1 stem loop within CR4/5 is not stably folded in the absence of the telomerase reverse transcriptase in vitro. Rather, the hTR CR4/5 domain adopts a heterogeneous ensemble of conformations. Finally, single-molecule FRET measurements of CR4/5 and a mutant designed to stabilize the P6.1 stem demonstrate that TERT-binding selects for a structural conformation of CR4/5 that is not the dominant state of the TERT-free in vitro RNA ensemble.

中文翻译：

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人类端粒酶 RNA 三向连接处的折叠异质性

端粒通过抑制染色体末端的非法 DNA 损伤反应来保护基因组。为了补偿端粒处不完整的 DNA 复制，大多数持续分裂的细胞，包括许多癌症，都会表达端粒酶核糖核蛋白 (RNP) 复合物。端粒酶通过使用内部端粒酶 RNA (TR) 模板催化短 DNA 重复序列的从头合成来维持端粒长度。来自不同物种的 TRs 具有结构上保守的域，有助于 RNP 生物发生和功能。在脊椎动物 TRs 中，保守区域 4 和 5 (CR4/5) 折叠成三路连接 (TWJ)，直接与端粒酶催化蛋白亚基结合，是端粒酶功能所必需的。我们使用体外化学作图的组合分析了人类 TR (hTR) CR4/5 结构域的结构特性，二级结构建模和单分子结构分析。我们的数据表明，在体外没有端粒酶逆转录酶的情况下，CR4/5 中必不可少的 P6.1 茎环不能稳定折叠。相反，hTR CR4/5 域采用了异构的构象集合。最后，CR4/5 的单分子 FRET 测量和旨在稳定 P6.1 茎的突变体表明，TERT 结合选择了 CR4/5 的结构构象，这不是无 TERT 体外 RNA 的主要状态合奏。