Structural studies of protein-nucleic acid interactions

描述(申请人提供)：在逆转录病毒的生命周期中，逆转录酶(RT)首先利用单链DNA合成DNA 以逆转录病毒RNA为模板，随后将互补的DNA链作为 一种模板，用于产生基因组的双链DNA拷贝。 与合成活性相结合的是RNaseH活性，它能降解 基因组RNA，并允许RT完成DNA第二链的合成。 然后将逆转录病毒基因组的双链DNA拷贝整合到 宿主的基因组通过整合酶，另一种逆转录病毒酶。 在成功整合到宿主基因组后，逆转录病毒 依靠宿主的机器来制作逆转录病毒的转录 基因组，这些基因组随后通过利用 一条现有的宿主核出口途径。核出口途径，即 被D型逆转录病毒使用，它编码一种被称为 构成运输元件(Cth)，是一种通常用于 输出mrna。介导m RNA和m RNA核输出的宿主蛋白 逆转录病毒RNA包括Cth is Tap。 拟议的研究包括x射线结晶学研究和相关的 旨在了解蛋白质-核酸的功能研究 原子细节上的相互作用需要以下几点：(1)启动 逆转录病毒复制和(2)未剪接逆转录病毒RNA的核输出 包含Cth的。这些研究更广泛地涉及(1) 对同一宿主介导的mRNA核输出的理解 因子Tap，以及(2)对核酸相互作用的理解 在复制过程中通过比较结构分析与 相关聚合酶。结构研究已经被提出用于生物学 与三种蛋白质形成的新型核酸复合体，包括 人蛋白TAP，Moloney鼠白血病病毒逆转录酶 Ri)，以及MMLV RT的N端片段。这些项目有一个共同的目标： 获得与源自CTE的RNA分子的络合物，其为 兴趣是一种新的RNA分子，除了它在 介导未剪接逆转录病毒RNA的核输出。由于CTE是一种 逆转录病毒成分，它必须通过逆转录酶和 因此，它直接与这种酶相互作用。这些研究提供了一种独特的 比较生物上相关的核酸相互作用的机会 不同蛋白质的CTH。

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.