Structure and Dynamics of the Telomerase Ribonucleoprotein

项目摘要 端粒酶核糖核蛋白(Rnp)是维持端粒这种特殊核蛋白所必需的。 保护真核细胞染色体末端免受异常加工和有害的端到端融合的结构 Sion事件。端粒酶通过独特的催化机制催化端粒DNA的进行性延伸 这就要求端粒酶RNA、端粒酶逆转录酶具有很强的功能相互依赖性 (TERT)，以及几个额外的蛋白质亚基。这项提议的主要目标是阐明 保守的结构RNA和蛋白质结构域协调端粒酶RNP的组装过程，催化- SIS和端粒招募。为了实现这一目标，我们将利用一种多方面的实验策略 将单分子生物物理技术与计算、生化和高分辨率相结合 结构性方法。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，我们将使用生化结构探测，单分子Förster共振能量 转移(SmFRET)和X射线结晶学分析端粒酶采用的结构中间态 RNP组装途径中的RNA和TERT。在目标2中，我们将使用smFRET和一种新型的端粒酶Ac- 活性检测方法研究驱动端粒的蛋白质和RNA结构域的构象动力学- ASE功能。在目标3中，我们将研究端粒酶的动态DNA处理特性。这些 实验将集中在端粒酶重新聚集到端粒以及端粒下的分子机制上。 端粒DNA的固有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.