HIGHLY SELECTIVE AFFINITY PROBES--DNA & RNA POLYMERASES

高选择性亲和探针--DNA

• 批准号: 3432697
• 负责人: Alexander Goldfarb
• 金额: $ 2万
• 依托单位: PUBLIC HEALTH RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-06-01 至 1996-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3432697
• 关键词: DNA; DNA directed DNA polymerase; DNA directed RNA polymerase; RNA; affinity labeling; chemical binding; crosslink; deoxyribonucleotides; molecular site; nucleic acid chemical synthesis; nucleic acid probes; nucleic acid sequence; nucleotide analog; ribonucleotides; site directed mutagenesis

This collaborative project is directed at designing, synthesizing and characterizing a new generation of crosslinkable derivatives of ribo and deoxyribonucleotides to be used for structural probing of a variety of polymerizing enzymes, including DNA and RNA polymerases of prokaryotic, eukaryotic and viral origin. The approach is based on the highly selective affinity labeling technique developed by the Russian colleagues participating in the collaboration. Two classes of compounds will be synthesized: (a) with reactive group positioned on the gamma-phosphate and (b) with substitutions in the base ring of uracil. In each class, crosslinking reagents specific to different amino acids, with different mechanisms of activation, and with varying length of the reagent "arm" will be prepared. Such types of nucleotide derivatives can be utilized as substrates during in vitro enzymatic syntheses of DNA and RNA. Thus, the reagents synthesized can be used for in vitro crosslinking to the substrate or product binding site of the polymerase under study followed by extension with the next radiolabeled nucleotide resulting in a radioactive nucleotide reporter group crosslinked to the protein. The site of the crosslinking can then be mapped using standard protein chemical approaches. This proposal represents an expansion of the currently active parent NIH grant (R0l GM30717) focused on site-directed mutagenesis of RNA polymerase genes in E. coli and functional analysis of the mutant enzymes. Mapping of functional sites using new affinity reagents will be invaluable for planning and interpreting the genetic experiments of the parent project.

该合作项目旨在设计、综合和 表征新一代可交联核糖衍生物和 脱氧核糖核苷酸可用于多种结构探测 聚合酶，包括原核生物的DNA和RNA聚合酶， 真核和病毒起源。 该方法基于高度 俄罗斯同事开发的选择性亲和标记技术 参与合作。 两类化合物将是 合成：(a) 反应基团位于 γ-磷酸酯上 (b)在尿嘧啶基环上有取代。 在每个班级中， 交联剂针对不同的氨基酸，具有不同的 激活机制，以及不同长度的试剂“臂” 将会做好准备。 可以利用此类类型的核苷酸衍生物 作为 DNA 和 RNA 体外酶促合成过程中的底物。 因此， 合成的试剂可用于体外交联 随后研究聚合酶的底物或产物结合位点 通过延伸下一个放射性标记的核苷酸，产生 放射性核苷酸报告基团与蛋白质交联。 这 然后可以使用标准蛋白质来绘制交联位点 化学方法。 该提案代表了对 目前活跃的父级 NIH 拨款 (R0l GM30717) 重点关注定点定向 大肠杆菌RNA聚合酶基因的诱变及其功能分析 突变酶。 使用新亲和力绘制功能位点 试剂对于规划和解释遗传基因将具有无价的价值。 父项目的实验。