M.V. Lomonosov Moscow State University, Moscow, 119991, Russia.
Skolkovo Institute of Science and Technology, Moscow, 121205, Russia.
Sci Rep. 2020 Jul 6;10(1):11109. doi: 10.1038/s41598-020-68107-x.
Telomerase is a ribonucleoprotein enzyme, which maintains genome integrity in eukaryotes and ensures continuous cellular proliferation. Telomerase holoenzyme from the thermotolerant yeast Hansenula polymorpha, in addition to the catalytic subunit (TERT) and telomerase RNA (TER), contains accessory proteins Est1 and Est3, which are essential for in vivo telomerase function. Here we report the high-resolution structure of Est3 from Hansenula polymorpha (HpEst3) in solution, as well as the characterization of its functional relationships with other components of telomerase. The overall structure of HpEst3 is similar to that of Est3 from Saccharomyces cerevisiae and human TPP1. We have shown that telomerase activity in H. polymorpha relies on both Est3 and Est1 proteins in a functionally symmetrical manner. The absence of either Est3 or Est1 prevents formation of a stable ribonucleoprotein complex, weakens binding of a second protein to TER, and decreases the amount of cellular TERT, presumably due to the destabilization of telomerase RNP. NMR probing has shown no direct in vitro interactions of free Est3 either with the N-terminal domain of TERT or with DNA or RNA fragments mimicking the probable telomerase environment. Our findings corroborate the idea that telomerase possesses the evolutionarily variable functionality within the conservative structural context.
端粒酶是一种核糖核蛋白酶，在真核生物中维持基因组完整性，并确保细胞的持续增殖。耐热酵母汉逊酵母中的端粒酶全酶除了含有催化亚基（TERT）和端粒酶 RNA（TER）外，还含有辅助蛋白 Est1 和 Est3，它们对端粒酶的体内功能至关重要。在这里，我们报告了汉逊酵母 Est3（HpEst3）在溶液中的高分辨率结构，以及其与端粒酶其他成分的功能关系的特征。HpEst3 的整体结构与酿酒酵母和人类 TPP1 的 Est3 相似。我们已经表明，汉逊酵母中的端粒酶活性以功能对称的方式依赖于 Est3 和 Est1 蛋白。缺少任何一种 Est3 或 Est1 都会阻止稳定的核糖核蛋白复合物的形成，削弱第二个蛋白与 TER 的结合，并减少细胞 TERT 的数量，这可能是由于端粒酶 RNP 的不稳定性。NMR 探测表明，游离的 Est3 与 TERT 的 N 端结构域或与模拟可能的端粒酶环境的 DNA 或 RNA 片段之间没有直接的体外相互作用。我们的发现证实了这样一种观点，即端粒酶在保守的结构背景下具有进化上可变的功能。
