REGULATORY MECHANISMS OF THE E COLI ARABINOSE OPERON

我们工作的长期目标是通过 大肠杆菌中的L-阿拉伯糖操纵子受哪些调控。这将是 有必要在我们对蛋白质结构、DNA的理解上取得进展 结构、蛋白质-DNA相互作用、蛋白质-蛋白质相互作用和细胞 生理学。 野生型、突变型和突变型转录的激活和抑制 基因工程的AraC蛋白将在体内和体外进行研究。 AraC蛋白的四聚化，当该蛋白形成DNA时发生 环，将通过凝胶电泳带移位分析进行研究，并 高效液相色谱检测方法将进一步发展和利用。Arac蛋白和两个 鼠李糖操纵子的结构类似物，ras和ras，将是 研究确定与DNA接触的亚基的氨基酸， 特定的氨基酸或区域是否可以被识别为激活 转录，为什么在某些情况下二聚体AraC蛋白会接触 三个相邻的DNA主要凹槽，在其他情况下接触四个 相邻的主要凹槽。 将对DNA环路进行调查。我们将通过体外转录进行测试 体外循环大小的上限和下限是否与我们的 已经在活体内发现。在体外不同的行为将表明 一种影响循环的蛋白质，可能是一种类似组蛋白的蛋白质。通过 改变AraC蛋白结合的序列，我们将扩展 观察到AraC蛋白结合的序列可能会影响 它的行为。我们还将检查对循环和规则的影响 在循环中引入一段弯曲序列，其效果 将一个AraC蛋白结合位点改变为另一个，并逆转 Arac蛋白结合位点。 我们将开发和测试一种遗传方法来定位地球表面区域 一种蛋白质。如果它对天门冬氨酸氨基转移酶有效，我们将应用它 并试图确定，以及，以及，区域 参与激活和四聚化的蛋白质。

The long-term objectives of our work are to understand the mechanisms by which the L-arabinose operons in Escherichia coli are regulated. This will necessitate advances in our understanding of protein structure, DNA structure, protein-DNA interactions, protein-protein interactions, and cell physiology. Activation and repression of transcription by wild type, mutant, and genetically engineered AraC protein will be studied in vivo and in vitro. Tetramerization of AraC protein, as occurs when the protein forms DNA loops, will be studied by gel electrophoresis band shift assays, and an HPLC assay will be further developed and utilized. AraC protein and two structural analogues from the rhamnose operon, RhaS and RhaS, will be studied to determine the amino acids of a subunit that contact the DNA, whether specific amino acids or regions can be identified as activating transcription, why the dimeric AraC protein in some instances contacts three adjacent major grooves of DNA, and in other instances contacts four adjacent major grooves. DNA looping will be investigated. We will test with in vitro transcription whether the same upper and lower limits to loop size exist in vitro as we have found in vivo. Different behavior in vitro would indicate the absence of a protein, perhaps a histone-like protein, that affects looping. By varying the sequences to which AraC protein binds, we will extend observations that the sequence to which AraC protein is bound can affect its behavior. We will also examine the effect on looping and regulation of introducing a stretch of bent sequence into the loop, the effects of changing one AraC protein binding site to another, and of inverting the AraC protein binding sites. We will develop and test a genetic method for locating surface regions of a protein. If it works with aspartate transcarbamoylase, we will apply it to AraC protein and attempt to determine, as well, the regions of the protein involved with activation and tetramerization.