STRUCTURAL STUDIES OF ENZYME/NUCLEIC ACID INTERACTIONS

酶-核酸相互作用代表了 需要结构和功能数据的研究 阐明一般原则。尽管有大量的 仅催化核酸底物反应的酶 已报道了12种酶-核酸复合体结构 约会。本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 通过逆转录酶(RT)聚合。相对的 RTS的简单性，保留了有效的复制能力，但缺乏 其他复制聚合酶的相关成分，使它们 是研究聚合过程的理想之选。 RTS结合了两种催化活性，即RNA或DNA导向的DNA 聚合酶活性和核糖核酸酶H(RNAseH)活性，顺序为 制作单链RNA基因组的双链DNA拷贝 逆转录病毒。然后，逆转录病毒DNA被整合到宿主体内 基因组的逆转录病毒整合酶，以及宿主的细胞机制 被用来制造更多的逆转录病毒。艾滋病的流行 由人类免疫缺陷病毒引起的L(艾滋病毒-L)已成为 HIV-1逆转录酶作为药物靶点的大量研究 心理治疗。 我们建议使用x射线结晶学方法来确定 含催化片断的核酸络合物的结构 Moloney鼠白血病病毒(MMLV)RT和-复合体与完整- 长度为MMLV RT。我们的方法是比较 MMLV RT和HIV-L RT的结构 酶起作用了。基本和详细的催化和底物知识 RT中的交互作用需要进一步努力才能有效地开发 抑制剂。从我们对MMLV RT-核酸复合体的分析 结构，我们建议阐明一个详细的催化机理。 对于RT聚合，确定重要的相互作用 核酸，并鉴定底物的结构基础 专一性。此外，我们计划进行定点突变。 特定氨基酸残基将被取代的研究 关于突变体RTS的结构分析和功能性质 将被描述为。晶体结构将被确定为 与核酸络合的有趣突变体RTS及其比较 野生型酶复合体。我们预计，从 这些研究将扩展到其他相关的聚合酶和我们的一般 关于酶-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.