DEOXYRIBONUCLEOTIDE METABOLISM IN ESCHERICHIA COLI

核糖核苷酸还原酶催化还原 从核糖核苷酸到脱氧核糖核苷酸。分离出的酶 已经得到了广泛的鉴定和鉴定 作为其他来源的核糖核苷酸还原酶的模型 消息来源。高等真核生物中的这种酶似乎非常 类似于大肠杆菌中的酶。一种单一的酶催化 所有核糖核酸二磷酸及其底物的还原 特异性和整体活性受变构控制 监管。酶的合成的调节是 不同寻常。核糖核苷酸编码基因的表达 还原酶(Nrd)似乎与DNA复制的控制平行。 因此，了解其调控的分子机制 NRD表达将是理解NRD的重要一步 DNA复制的调控。这反过来又将是一个重大的 了解细胞生长控制的一步。 因为这种酶的水平与细胞生长成正比 无论是真核生物还是在大肠杆菌中，这种酶似乎都是一种 抗肿瘤化疗药物的理想靶点 细胞。由于这种酶是由疱疹病毒和Epstein-Bar编码的 病毒，它可能成为化疗药物的靶点 对抗这些病毒。 为了了解NRD调控的分子细节， 到目前为止，实验已经利用胸腺嘧啶剥夺来改变NRD 表情。这项提案的一个目标是调查NRD 在指数生长细胞中的表达作为细胞的函数 周而复始。初步实验表明，NRD的表达 在胸腺嘧啶剥夺期间观察到的结果是由于 通常发生在细胞周期中。进一步调查 NRd调控的细节，含有NRd的质粒 融合到LacZ的监管区域将被用来分离和 描述交易突变体的特征。这些突变体将被用来 克隆并鉴定“野生型”等位基因。一种DNA蛋白质 结合聚丙烯酰胺凝胶试验将用于鉴定和 纯化调节蛋白。将使用DNA酶足迹来 表明这些蛋白质结合到被鉴定为操纵子的位置 网站。体外产生调控区5‘端的点突变 将对nrd的结构基因进行鉴定和测序。 以进一步定义涉及正面和负面的站点 监管。

Ribonucleotide reductases catalyze the reduction of ribonucleotides to deoxyribonucleotides. The enzyme isolated from Escherichia coli has been extensively characterized and serves as the model for ribonucleotide reductases from other sources. The enzyme in higher eucaryotes appears to be very similar to the E. coli enzyme. A single enzyme catalyzes the reduction of all ribonucleotide diphosphates and the substrate specificity and overall activity is controlled by allosteric regulation. The regulation of the synthesis of the enzyme is unusual. The expression of the genes encoding ribonucleotide reductase (nrd) appear to parallel the control of DNA replication. Thus, understanding the molecular mechanism of the control of nrd expression will be an important step in the understanding of the regulation of DNA replication. This in turn will be a major step in understanding the control of cell growth. Since the level of this enzyme is proportional to cell growth in both eucaryotes and in E. coli, this enzyme would appear to be an ideal target for chemotherapeutic agents active against tumor cells. Since this enzyme is encoded by Herpes and Epstein-Bar virus, it could be a possible target for chemotherapeutic agents against these viruses. To understand the molecular details of nrd regulation, experiments to date have utilized thymine deprivation to alter nrd expression. One objective of this proposal is to investigate nrd expression in exponentially growing cells as a function of the cell cycle. Preliminary experiments suggest that the nrd expression observed during thymine deprivation results from regulation normally occurring during the cell cycle. To further investigate the details of nrd regulation, plasmids containing the nrd regulatory region fused to lacZ will be utilized to isolate and characterize transacting mutants. These mutants will be used to clone and characterize the "wild type" allele. A DNA-protein binding polyacrylamide gel assay will be used to identify and purify the regulatory proteins. DNase footprinting will be used to show that these proteins bind to the sites identified as operator sites. In vitro generated point mutants in the regulatory region 5' to the structural genes of nrd will be characterized and sequenced to further define sites involved in both positive and negative regulation.