STRUCTURE/FUNCTION STUDIES OF HIV-1 RT

核酸合成的启动是生物进化过程中的关键步骤。 遗传信息的复制和转录。一个详细的 对这一过程的分子描述将对 广泛的领域，从抗病毒药物的设计到 癌细胞增殖的调控。我建议研究一下 人类免疫缺陷病毒1型(HIV-1)启动DNA合成 1)从tRNA引物退火到RNA的逆转录酶(RT 模板，一个建立在广泛的结构、生化、 分子遗传信息已经可用。HIV-1的晶体 RT与单独的tRNA和退火短模板的tRNA络合 RNA是在托马斯·斯泰茨博士的实验室里获得的，并且 绕射到中等分辨率。为了获得结晶学经验， 我将加入正在进行的解决这些结构的工作。来补充 扩展这些研究，我计划系统地改变这两种蛋白质 和起始复合体的核酸成分，目标是 获得衍射率更高的晶体。具体来说，我 将(1)结晶并确定蛋白质-核酸的结构 参与HIV-1RT启动DNA合成的复合体，以及 (2)体外评价这些复合体的功能重要性 启蒙试验。我希望通过这些来解决的问题 研究包括：在RT和RNA之间有什么联系 形成了一个启动复合体？TRNA引物是如何解开的 对模板RNA进行退火热处理？RT中发生了哪些构象变化 在DNA合成的启动过程中？这些问题的答案将 为聚合酶的作用机制提供了有价值的见解 合成核酸。

Initiation of nucleic acid synthesis is a critical step in the replication and transcription of genetic information. A detailed molecular description of this process will be important for research in a wide variety of fields, ranging from the design of antiviral drugs to the regulation of cancer cell proliferation. I propose to study the initiation of DNA synthesis by human immunodeficiency virus type 1 (HIV- 1) reverse transcriptase (RT) from a tRNA primer annealed to an RNA template, a project that builds on the extensive structural, biochemical, and molecular genetic information already available. Crystals of HIV-1 RT complexed with tRNA alone and with tRNA annealed to a short template RNA have been obtained in the laboratory of Dr. Thomas Steitz, and diffract to moderate resolution. To gain crystallographic experience, I will join the ongoing work to solve these structures. To complement and extend these studies, I plan to systematically alter both the protein and nucleic acid components of the initiation complex, with the goal of obtaining crystals that diffract to higher resolution. Specifically, I will (1) crystallize and determine the structure of protein-nucleic acid complexes involved in the initiation of DNA synthesis by HIV-1 RT, and (2) evaluate the functional importance of these complexes using in vitro assays for initiation. Questions that I hope to address through these studies include: What contacts are made between RT and RNA during the formation of an initiation complex? How is the tRNA primer unwound and annealed to the template RNA? What conformational changes occur in RT during the initiation of DNA synthesis? Answers to these questions will provide valuable insight into the mechanisms by which polymerases synthesize nucleic acids.