Structure and Dynamics of the Telomerase Ribonucleoprotein

项目摘要 端粒酶核糖核蛋白（RNP）是维持端粒所必需的， 保护真核细胞染色体末端免受异常加工和有害的端对端融合的结构， 锡永。端粒酶通过一种独特的催化机制催化端粒DNA的进行性延伸 这需要端粒酶RNA，端粒酶逆转录酶， （TERT）和几个额外的蛋白质亚基。本提案的主要目的是阐明如何 保守的结构RNA和蛋白质结构域协调端粒酶RNP组装的过程， 姐妹和端粒的募集。为了实现这一目标，我们将利用多方面的实验策略， 结合了单分子生物物理技术与计算，生物化学和高分辨率 结构方法。我们将研究人类端粒酶和四膜虫模型系统中的酶 嗜热菌在目标1中，我们将采用生物化学结构探测，单分子Förster共振能量 转移（smFRET）和X射线晶体学来分析端粒酶所采用的结构中间状态 RNA和TERT在RNP装配途径中的作用。在目标2中，我们将使用smFRET和一种新的端粒酶活性， 动力学检测方法，以研究驱动端粒的蛋白质和RNA结构域的构象动力学， ase函数目的3：研究端粒酶的动态DNA处理特性。这些 实验将集中在端粒酶募集端粒的分子机制，以及在- 了解端粒DNA的内在折叠特性如何调节端粒酶催化。

Project Summary The telomerase ribonucleoprotein (RNP) is required for maintaining telomeres, the specialized nucleoprotein structures that protect eukaryotic chromosome ends from aberrant processing and deleterious end-to-end fu- sion events. Telomerase catalyzes processive extension of telomere DNA via a unique catalytic mechanism that requires a strong functional interdependence of the telomerase RNA, telomerase reverse transcriptase (TERT), and several additional protein subunits. The primary objective of this proposal is to elucidate how conserved structural RNA and protein domains coordinate the processes of telomerase RNP assembly, cataly- sis, and recruitment to telomeres. To address this goal, we will utilize a multi-faceted experimental strategy that combines single-molecule biophysical techniques paired with computational, biochemical, and high-resolution structural approaches. We will study human telomerase and the enzyme from the model system Tetrahymena thermophila. In aim 1, we will employ biochemical structure probing, single-molecule Förster resonance energy transfer (smFRET), and x-ray crystallography to analyze structural intermediate states adopted by telomerase RNA and TERT during the RNP assembly pathway. In aim 2, we will use smFRET and a novel telomerase ac- tivity detection method to investigate conformational dynamics of protein and RNA domains that drive telomer- ase function. In aim 3, we will study the dynamic DNA handling properties of the telomerase enzyme. These experiments will focus on the molecular mechanisms for telomerase recruitment to telomeres as well as under- standing how the intrinsic folding properties of telomere DNA regulate telomerase catalysis.