Structural studies of protein-nucleic acid interactions

描述（由申请人提供）：在逆转录病毒生命周期中，逆转录病毒 转录酶（RT）的合成DNA首先使用单链的 逆转录病毒RNA作为模板，随后互补DNA链作为模板， 一个模板，以产生基因组的双链DNA拷贝。 与合成活性偶联的是RNA酶H活性， 它可以通过逆转录酶完成第二条DNA链的合成。 然后将逆转录病毒基因组的双链DNA拷贝整合到 整合酶，另一种逆转录病毒酶。 在成功整合到宿主基因组中后，逆转录病毒然后 依靠宿主的机制来转录逆转录病毒 基因组，随后从细胞核中输出， 现有的核出口通道核出口途径， 由D型逆转录病毒使用，其编码被称为 组成型转运元件（CTh）是通常用于 输出mRNA。介导mRNA核输出的宿主蛋白， 包括CTh的逆转录病毒RNA是Tap。 拟议的研究包括x射线晶体学研究和相关的 功能性研究，旨在了解蛋白质-核酸 原子细节的相互作用需要以下内容：（1）启动 逆转录病毒复制和（2）未剪接的逆转录病毒RNA的核输出 含有CTh。这些研究更普遍地涉及（1） 理解由同一宿主介导的mRNA的核输出 因子Tap，以及（2）理解核酸相互作用， 通过比较结构分析， 相关聚合酶结构研究已被提议用于生物学 相关的和新的核酸复合物与三种蛋白质，包括 人蛋白Tap，莫洛尼鼠白血病病毒逆转录酶（MMLV RI）和MMLV RT的N-末端片段。这些项目有一个共同的目标， 获得具有衍生自CTE的RNA分子的复合物，其为 作为一种新RNA分子，除了其在以下方面的生物学作用之外， 介导未剪接的逆转录病毒RNA的核输出。因为CTE是一个 逆转录病毒元件，它必须通过逆转录酶复制， 因此直接与这种酶相互作用。这些研究提供了一个独特的 有机会比较生物学相关的核酸相互作用的 CTh与不同的蛋白质。

DESCRIPTION (provided by applicant): During the retroviral life cycle, reverse transcriptase (RT) processively synthesizes DNA using first the single-stranded retroviral RNA as the template and subsequently the complementary DNA strand as a template in order to produce a double-stranded DNA copy of the genome. Coupled to the synthetic activities is an RNase H activity that degrades the genomic RNA and allows RT to complete synthesis of the second strand of DNA. The double-stranded DNA copy of the retroviral genome is then integrated into the host's genome by integrase, another retroviral enzyme. Following successful integration into the host genome, the retrovirus then relies on the host's machinery in order to make transcripts of the retroviral genome, which are subsequently exported from the nucleus by taking advantage of an existing host nuclear export pathway. The nuclear export pathway that is used by type D retroviruses, which encode an RNA element referred to as the constitutive transport element (CTh), is a pathway that is normally used to export mRNA. The host protein that mediates nuclear export of mRNA and retroviral RNA including the CTh is Tap. The proposed studies include x-ray crystallographic studies and related functional studies that are directed toward understanding protein-nucleic acid interactions in atomic detail required for the following: (1) the initiation of retroviral replication and (2) nuclear export of unspliced retroviral RNA containing the CTh. These studies are related more generally to (1) the understanding of nuclear export of mRNA, which is mediated by the same host factor Tap, and (2) the understanding of nucleic acid interactions that are important during replication through comparative structural analyses with related polymerases. Structural studies have been proposed for biologically relevant and novel nucleic acid complexes with three proteins including the human protein Tap, Moloney murine leukemia virus reverse transcriptase (MMLV RI), and an N-terminal fragment of MMLV RT. The projects share a common goal of obtaining a complex with an RNA molecule derived from the CTE, which is of interest as a novel RNA molecule in addition to its biological role in mediating nuclear export of unspliced retroviral RNA. As the CTE is a retroviral element, it must be replicated by reverse transcriptase and therefore interact directly with this enzyme. These studies provide a unique opportunity to compare biologically relevant nucleic acid interactions of the CTh with different proteins.