STRUCTURAL STUDIES OF ENZYME/NUCLEIC ACID INTERACTIONS

酶-核酸相互作用代表了生物学的一个基本领域， 需要结构和功能数据的研究，以便 阐明一般原则。虽然有大量的 仅催化核酸底物反应的酶， 已经报道了12种酶-核酸复合物结构， 约会该提案描述了结构和功能研究 旨在解决酶-核酸相互作用的重要性 逆转录酶（RT）聚合。的相对 保留有效复制能力，但缺乏 其他复制聚合酶的相关成分，使它们 非常适合研究聚合过程。 RT偶联两种催化活性，RNA或DNA指导的DNA 聚合酶活性和核糖核酸酶H（RNaseH）活性， 复制出单链RNA基因组的双链DNA 逆转录酶病毒然后逆转录病毒DNA被整合到宿主体内 逆转录病毒整合酶和宿主的细胞机器 用来生产更多的逆转录病毒。艾滋病的流行 由人类免疫缺陷病毒1型（HIV-1）引起的艾滋病已经成为人们关注的焦点。 作为药物靶点的HIV- 1 RT的大量研究工作 疗法 我们建议使用X射线晶体学方法来确定 具有催化片段的核酸复合物的结构 莫洛尼鼠白血病病毒（MMLV）RT和-复合物的完整- 长度MMLV RT。我们的方法是比较保守的功能， MMLV RT和HIV-1 RT的结构，以便了解 酶起作用。催化和底物的基本和详细知识 RT中的相互作用将进一步努力开发有效的 抑制剂的根据我们对MMLV RT-核酸复合物的分析， 结构，我们建议阐明一个详细的催化机制 对于RT聚合，以确定与 核酸，并确定底物的结构基础 的特异性此外，我们计划进行定点突变， 其中特定氨基酸残基将被取代的研究 我们的结构分析和功能特性的突变的RT 将被定性。晶体结构将被确定为 与核酸复合的有趣的突变RT，并进行比较 与野生型酶复合物的关系我们希望， 这些研究将扩展到其他相关的聚合酶和我们的一般 酶与核酸相互作用的知识。

Enzyme-nucleic acid interactions represent a fundamental area of research that requires structural and functional data in order to elucidate general principles. Although there are a large number of enzymes which catalyze reactions for nucleic acid substrates, only twelve enzyme-nucleic acid complex structures have been reported to date. This proposal describes structural and functional studies designed to address the importance of enzyme-nucleic acid interactions in polymerization by reverse transcriptase (RT). The relative simplicity of RTs, which retain effective replicative capacity but lack the associated components of other replicative polymerases, make them ideal for studying the process of polymerization. RTs couple two catalytic activities, an RNA- or DNA-directed DNA polymerase activity and a ribonuclease H (RNaseH) activity, in order to make a double-stranded DNA copy of the single-stranded RNA genome of the retrovirus. The retroviral DNA is then integrated into the host genome by the retroviral integrase, and the host's cellular machinery is utilized to produce more retrovirus. The epidemic outbreak of AIDS caused by human immunodeficiency virus type l (HIV- l) has focused a great deal of research efforts on HIV- 1 RT as a target for drug therapy. We propose to use x-ray crystallographic methods to determine the structures of a nucleic acid complex with a catalytic fragment of Moloney murine leukemia virus (MMLV) RT and- complex with the full- length MMLV RT. Our apprdach is to compare conserved features of the structures of MMLV RT and HIV- l RT in order to understand how the enzyme works. Basic and detailed knowledge of catalysis and substrate interactions in RT wlll further efforts in the development of effective inhibitors. From our analyses of the MMLV RT-nucleic acid complex structures, we propose to elucidate a detailed mechanism of catalysis for polymerization by RT, to determine important interactions with nucleic acid, and to identify the structural basis of substrate specificity. In addition, we plan to pursue site-directed mutagenesis studies in which specific amino acid residues will be substituted based on our structural analyses, and functional properties of the mutant RTs will be characterized. Crystal structures will be determined for interesting mutant RTs complexed with nucleic acid and comparisons made to the wild-type enzyme complex. We expect that insights derived from these studies will extend to other related polymerases and our general knowledge of enzyme-nucleic acid interactions.