STRUCTURE/FUNCTION STUDIES OF HIV-1 RT

核酸合成的起始是核酸合成中的关键步骤。 遗传信息的复制和转录。 详细 这一过程的分子描述将是重要的研究， 广泛的领域，从抗病毒药物的设计， 调节癌细胞增殖。 我建议研究 人类免疫缺陷病毒1型（HIV-1）启动DNA合成 1)来自退火至RNA的tRNA引物的逆转录酶（RT） 模板，一个建立在广泛的结构，生物化学， 和分子遗传学信息。 HIV-1的晶体 RT与单独的tRNA和与短模板退火的tRNA复合 RNA已在托马斯施泰茨博士的实验室获得， 分辨率为中等。 为了获得晶体学的经验， 我将加入正在进行的工作，以解决这些结构。 以补充 并扩展这些研究，我计划系统地改变蛋白质， 和起始复合物的核酸组分，目的是 获得了具有更高分辨率的晶体。 我特别 将（1）结晶并确定蛋白质-核酸的结构 参与HIV-1 RT启动DNA合成的复合物，以及 (2)评价这些复合物的功能重要性， 启动试验。 我希望通过这些来解决的问题 研究包括：RT和RNA之间的接触是什么， 一种起始复合体的形成 tRNA引物是如何解开的， 退火到模板RNA上？ RT发生了什么构象变化 在DNA合成的初始阶段 这些问题的答案将 提供了有价值的洞察机制， 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.