Structural Study of Vertebrate Telomerase RNA

描述(申请人提供)：端粒酶是一种核糖核蛋白复合体，催化将端粒DNA重复序列添加到线性染色体的3‘端。端粒保护染色体末端不被细胞DNA修复机制识别为双链断裂，也防止末端连接。在没有活性端粒酶的情况下，端粒会随着每一轮DNA复制而缩短。一旦丢失了临界数量的端粒，细胞就会进入复制性衰老，有时还会发生凋亡。大多数癌症中存在的高水平端粒酶活性可以避免这一障碍，使端粒酶成为潜在的药物靶点。端粒酶RNA和端粒酶逆转录酶是体外端粒酶活性的基本元件。这项研究的目标将是通过核磁共振技术解决青竹端粒酶RNAP2ab在催化核心中的高分辨结构，研究人端粒酶RNAP2ab作为抑制端粒酶活性的药物靶点的潜力，并获得青竹端粒酶RNA的小角X射线散射结构，包括游离的和与端粒酶逆转录酶复合的结构。本实验的具体目的是：(1)研究青竹端粒酶RNAP2ab结构域的结构、动力学和功能；(2)作为抑制端粒酶活性的药物靶点；(3)利用核磁共振和小角X射线散射技术建立青竹端粒酶RNA模型；(4)利用小角X射线散射和X射线结晶学方法研究青竹端粒酶RNA与端粒酶逆转录酶结构域之间的相互作用。这些实验将揭示青竹端粒酶RNA催化核心的结构，以及RNA和端粒酶逆转录酶亚域之间的分子间联系。

DESCRIPTION (provided by applicant): Telomerase is the ribonucleoprotein complex that catalyzes the addition of telomere DNA repeats onto the 3' ends of linear chromosomes. Telomeres protect the ends of chromosomes from being recognized by the cellular DNA repair machinery as double strand breaks and also prevent end joining. In the absence of active telomerase, telomeres shorten with each round of DNA replication. Once a critical amount of telomere is lost, the cell enters replicative senescence and sometimes undergoes apoptosis. The high level of telomerase activity present in most cancers allows this roadblock to be avoided, making telomerase a potential drug target. Telomerase RNA and telomerase reverse transcriptase comprise the essential elements for telomerase activity in vitro. The goal of this fellowship will be to solve the high- resolution structure of medaka telomerase RNA P2ab in the catalytic core by NMR, investigate a potential of human telomerase RNA P2ab as a drug target to inhibit telomerase activity, and obtain small angle X- ray scattering structures of the medaka telomerase RNA both free and in complex with the telomerase reverse transcriptase. The specific aims are to (1) investigate the structure, dynamics, and function of medaka telomerase RNA P2ab domain, (2) investigate human telomerase RNA J2a/b as a drug target to inhibit telomerase activity, (3) model medaka telomerase RNA using NMR and small angle X-ray scattering, (4) probe the structure and interactions between the medaka telomerase RNA and domains of the telomerase reverse transcriptase using small angle X-ray scattering and X-ray crystallography. These experiments will reveal the structure of the medaka telomerase RNA catalytic core and the intermolecular contacts between the RNA and the subdomain of the telomerase reverse transcriptase.