NUCLEASE ACTIVITY OF 1,10-PHENANTHROLINE COPPER ION

描述：(改编自申请人的摘要)建议的目标 研究包括：1)设计抑制核糖核酸的类似物 通过杂交到RNA合成的起始点进行转录；2)使用 调节蛋白-1，10-邻菲咯啉嵌合体用于鉴定未发现的DNA 基因组DNA中的结合序列。这两个研究方向都源于Dr。 Sigman对化学核酸酶活性的继续研究 1，10-二氮杂菲-铜(OP-Cu)。 在转录起始点形成的瞬间解开的DNA可以是 与模板互补的短核苷酸类似物杂交 一串串的发起人。对UV5和trpEDCBA启动子的研究将是 通过考察开放络合物作为以下函数的稳定性来扩展 核苷三磷酸浓度，化学修饰的骨架，以及 糖份。这些研究的目的将是a)确定无障碍的 该瞬时中间体的模板链内的序列，以及b)至 提高寡核苷酸的亲和力和启动子的特异性 抑制剂。真核生物开放复合体与真核生物开放复合体的同源性 原核生物的研究将特别注意三磷酸腺苷在 使单链DNA在真核系统中变得可用。核糖核酸 聚合酶辅助形成未缠绕的DNA使Short的使用 寡核苷酸可能用于基因特异性抑制物的设计 潜在的药理意义。 调控蛋白-OP嵌合体的定点切割允许 蛋白质与核酸结合的体外分析。定向删节也是 提供了一种识别调控序列的结合位点的方法 在基因组DNA中。一种检测这些断裂点的分析方法 设计出了一种能够识别大肠杆菌中的位点和 酵母基因组。此外，制备OP-嵌合体的新方法是基于 提出了OP-铜分离蛋白质的化学原理。这些方法 寻找调控基序将用大肠杆菌色氨酸抑制物和 GAL4、PUT3和PPR1的cAMP结合蛋白和锌簇蛋白 酵母。这些方法可以帮助描述非编码区的基因组。

DESCRIPTION: (adapted from applicant's abstract) The goals of the proposed research are 1) to design ribooligonucleotide analogs which inhibit transcription by hybridizing to initiation sites of RNA synthesis; 2) to use regulatory protein-1,10-phenanthroline chimeras to identify undiscovered DNA binding sequences in genomic DNAs. Both research directions stem from Dr. Sigman's continuing studies on the chemical nuclease activity of 1,10-phenanthroline-copper (OP-Cu). The transiently unwound DNA formed at transcription start sites can be hybridized by short ribooligonucleotide analogs complementary to the template strands of promoters. The studies with the UV5 and trpEDCBA promoters will be extended by examining the stability of the open complexes as a function of nucleoside triphosphate concentrations, chemically modified backbones, and sugar moieties. The aims of these studies will be a) to identify the accessible sequences within the template strand of this transient intermediate, and b) to increase the affinity and promoter specificity of the oligonucleotide inhibitors. The homology between the open complexes of eukaryotes and prokaryotes will be investigated with special attention to the role of ATP in making single-stranded DNAs accessible in eukaryotic systems. The RNA polymerase assisted formation of unwound DNA makes the use of short oligonucleotides possible in the design of gene specific inhibitors of potential pharmacological significance. The site-specific scission of regulatory protein-OP chimeras permits the analysis of nucleic acid binding by proteins in vitro. Targeted scission also provides an approach for identifying the binding sites of regulatory sequences in genomic DNA. An analytical method for detecting these sites of scission has been devised which would allow the identification of sites in the E. coli and yeast genomes. In addition, new methods of preparing OP-chimeras based on the chemistry of scission of proteins by OP-Cu have been devised. These methods of finding regulatory motifs will be tested with the E. coli trp repressor and cAMP binding proteins and the zinc cluster proteins of GAL4, PUT3 and PPR1 of yeast. These methods could help characterize genomes in non-coding regions.