REGULATORY MECHANISMS OF THE E COLI ARABINOSE OPERON

我们工作的长期目标是通过以下方式了解这些机制： 大肠杆菌中L-阿拉伯糖操纵子的调控。 这将 我们对蛋白质结构，DNA， 结构、蛋白质-DNA相互作用、蛋白质-蛋白质相互作用和细胞 physiology. 野生型、突变体和 基因工程AraC蛋白将在体内和体外进行研究。 AraC蛋白的四聚化，如蛋白质形成DNA时发生的 环，将通过凝胶电泳带移分析进行研究， 将进一步开发和使用HPLC测定法。 AraC蛋白和两种 来自鼠李糖操纵子的结构类似物RhaS和RhaS将被 研究以确定与DNA接触的亚基的氨基酸， 特定的氨基酸或区域是否可以被鉴定为激活 转录，为什么二聚体AraC蛋白在某些情况下接触 三个相邻的DNA大沟，在其他情况下， 相邻的主要凹槽。 将研究DNA循环。 我们将用体外转录来测试 体外是否存在与我们相同的环尺寸上限和下限 在体内发现的。 在体外的不同表现表明 一种蛋白质，也许是一种类似组蛋白的蛋白质，它影响着循环。 通过 改变AraC蛋白结合的序列，我们将延伸 观察到AraC蛋白结合的序列可以影响 它的行为。 我们还将研究对循环和调节的影响 在循环中引入一段弯曲的序列， 将一个AraC蛋白结合位点改变为另一个，并将AraC蛋白结合位点反转， AraC蛋白结合位点。 我们将开发和测试一种遗传方法，用于定位 蛋白质。 如果它与天冬氨酸转氨甲酰酶一起工作， AraC蛋白，并试图确定，以及， 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.