REGULATION OF ARABINOSE OPERONS IN ESCHERICHIA COLI

长期目标是更好地理解如何 调控蛋白相互作用并与DNA相互作用以控制基因 在原核生物中表达。大肠杆菌中的四个操纵子涉及 在对L的同化中，将对阿拉伯糖进行研究。三个具体的 问题将会得到解答。(1)araBAD，Arae的转录情况如何 和araFG操纵子被AraC蛋白和环-AMP受体激活 蛋白质？(2)AraC蛋白如何抑制操纵子的转录 当结合在DNA位点上时，启动子区域需要 诱导？(3)有哪些特定的蛋白质和DNA元素参与 ARAC对DNA结合位点的识别？ DNA聚合酶与DNA聚合酶之间特定接触点的位置 启动子DNA将通过DNA化学预修饰进行体外测定 技术采用凝胶电泳法进行DNA结合分析。先前 未检测到的启动子序列仅在中期阶段需要 结合过程也可以用这种技术来揭示。站点定向 用寡核苷酸进行突变将被用来测试体内的功能 具体的联系地点。 将对RNA聚合酶与每个 使用流产启动试验的启动子以及使用更直接的 凝胶电泳法-DNA结合试验。有了这些数据，个人 AraC和Cyclc-AMP受体蛋白在刺激中的作用 启动子结合可以根据初始结合和 活性“开放复合体”的异构化速率。推动者 结构-功能关系可以通过关联结合来研究 启动子在相对位置上存在差异的参数 激活剂。调控蛋白和启动子中的突变体 这些站点将用于体外和体内研究。Arac突变体 在激活特定启动子方面存在缺陷将在 研究蛋白质之间的相互作用和研究蛋白质相互作用的机制 来自上游DNA站点的抑制。 Arac蛋白结合了两类明显的DNA位点，它们可以是 以DNA上蛋白质接触区的范围来区分。 ARAC突变体将被选择结合一个，但不能结合另一个 地点。这些和在AraC和DNA结合位点上的额外突变 将有助于确定参与结合的蛋白质结构域。这个 突变体还应提供有关DNA位点对称性的信息。

The long range objective is to provide a better understanding of how regulatory proteins interact with each other and with DNA to control gene expression in procaryotes. The four operons in Escherichia coli involved in the assimilation of L-arabinose will be studied. Three specific questions will be addressed. (1) How is transcription of the araBAD, araE and araFG operons activated by both AraC protein and cyclic-AMP receptor protein? (2) How does AraC protein repress transcription of the operons when bound at DNA sites well upstream of the promoter regions required for induction? (3) What are the specific protein and DNA elements involved in AraC recognition of DNA binding sites? The locations of specific contact points between DNA polymerase and promoter DNA will be determined in vitro by DNA chemical premodification techniques using the gel electrophoresis DNA-binding assay. Previously undetected promoter sequences required only during intermediate stages of the binding process also can be revealed with this technique. Site-directed mutagenesis with oligonucleotides will be used to test the function in vivo of specific contact sites. Kinetic measurements will be made for RNA polymerase binding to each of the promoters using the abortive initiation assay as well using the more direct gel electrophoresis-DNA binding assay. With these data the individual contributions of AraC and cycli c-AMP receptor protein in stimulating promoter binding can be assessed in terms of the initial binding and the rate of isomerization to the active "open complex." Promoter structure-function relationships can be studied by correlating binding parameters with differences among the promoters in the relative positions of the activators. Mutants in the regulatory proteins and in the promoter sites will be used for studies in vitro and in vivo. AraC mutants defective in activating particular promoters will be especially useful in studying protein-protein interactions and in studying the mechanism of repression from upstream DNA sites. AraC protein binds two apparent classes of DNA sites that can be distinguished by the extent of the protein contact region along the DNA. AraC mutants will be selected that bind one, but fail to bind another site. These and additional mutations in AraC and in the DNA binding sites will aid in determining the protein domains involved in binding. The mutants should also provide information on the symmetry of the DNA sites.