NUCLEASE ACTIVITY OF 1,10-PHENANTHROLINE COPPER ION

描述：（改编自申请人的摘要） 研究是1）设计核糖寡核苷酸类似物，其抑制 通过与RNA合成的起始位点杂交进行转录; 2）使用 用调节蛋白-1，10-菲咯啉嵌合体鉴定未发现的DNA 基因组DNA中的结合序列。这两个研究方向都来自博士。 Sigman对化学核酸酶活性的持续研究 1，10-菲咯啉-铜（OP-Cu）。 在转录起始位点形成的瞬时解绕DNA可以是 通过与模板互补的短核糖寡核苷酸类似物杂交 启动子链。使用UV 5和trpEDCBA启动子的研究将在下文进行。 通过检查开放复合物的稳定性， 核苷三磷酸浓度，化学修饰的骨架，和 糖部分。这些研究的目的是：a）确定可获得的 该瞬时中间体的模板链内的序列，和B） 增加寡核苷酸的亲和力和启动子特异性 抑制剂的真核生物开放复合体与 原核生物将进行调查，特别注意ATP的作用， 使单链DNA在真核系统中可及。的RNA 聚合酶辅助的解绕DNA的形成利用短的 寡核苷酸可能在基因特异性抑制剂的设计， 潜在的药理学意义。 调节蛋白-OP嵌合体的位点特异性切割允许 体外蛋白质与核酸结合的分析。定向切割还 提供了一种鉴定调控序列结合位点的方法 基因组DNA中。检测这些断裂位点的分析方法， 设计了一个可以识别E.杆菌和 酵母基因组此外，还提供了基于本发明的制备OP-嵌合体的新方法。 已经设计出了通过OP-Cu切割蛋白质的化学。的这些方法 寻找调控基序将用E. colitrp阻遏物和 cAMP结合蛋白和锌簇蛋白GAL 4、PUT 3和PPR 1的表达， 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.