DIMERIZATION & COOPERATIVITY IN PROKARYOTIC GENE CONTROL

转录的控制依赖于DNA结合的调节蛋白及其相互作用。这项拟议的研究旨在使用几个原核模型系统来检验两类重要的蛋白质-蛋白质相互作用，即二聚化和协作性。遗传方法将被用来确定控制这些相互作用的特异性的分子决定因素。原核生物和真核生物中的许多转录调控因子都以二聚体的形式发挥作用，要么作为同源二聚体，要么作为异源二聚体，在某些情况下，相同的蛋白质可以与不同的伙伴结合，根据其伙伴执行不同的调节功能。因此，适当的生物活性取决于潜在伴侣相互区分的能力，从而只形成正确的二聚体物种。这项拟议的研究将通过分析大肠杆菌cAMP受体蛋白(CRP)及其同源物FNR的二聚化来解决二聚化特异性的问题。许多转录调节因子也以同型或异型组合的形式与DNA协同结合。特别是，真核调控复合体的组装通常涉及多个DNA结合的蛋白质，这些蛋白质参与各种弱的蛋白质-蛋白质相互作用。协作性既是一种通过提供陡峭的剂量-反应曲线来产生敏感的生物开关的机制，也是一种整合多种调节输入的手段。这项拟议的研究将通过对模型协作性界面、噬菌体Lambda阻遏物及其同系物的详细分析来探索潜在蛋白质-蛋白质相互作用的本质。这项研究的总体目标是开发可广泛应用于生物上重要的蛋白质-蛋白质相互作用研究的新的遗传方法。最近开发的一种基于大肠杆菌的双杂交试验将测试其在检测复杂文库中编码的相互作用蛋白方面的有效性。本实验还将应用于真核转录因子CREB、其CREB及其共激活因子CBP的磷酸化依赖的蛋白质相互作用的研究和操作。

The control of transcription depends on DNA-bound regulatory proteins and their interactions. The proposed research is aimed at examining two important classes of protein-protein interactions, dimerization and cooperativity, using several prokaryotic model systems. Genetic methods will be employed to identify the molecular determinants that govern the specificity of these interactions. Many transcriptional regulators in both prokaryotes and eukaryotes function in dimeric form, either as homodimers or heterodimers, and in some cases the same protein can associate with alternative partners, carrying out distinct regulatory functions depending on its partner. Thus, the proper biological activity depends on the abilities of potential partners to discriminate amongst one another so that only the correct dimeric species are formed. The proposed research will address the problem of dimerization specificity with an analysis of the dimerization specified of the E. coli cAMP receptor protein (CRP) and its homologue FNR. Many transcriptional regulators also bind cooperatively to the DNA in either homotypic or heterotypic combinations. The assembly of eukaryotic regulatory complexes, in particular, typically involves multiple DNA- bound proteins that participate in a variety of weak protein-protein interactions. Cooperativity is both a mechanism for generating sensitive biological switches by providing a steep dose-response cure, as well as a means by which multiple regulatory inputs can be integrated. The proposed research will probe the nature of the underlying protein- protein interactions with a detailed analysis of model cooperativity interface, that of the bacteriophage lambda repressor and its homologues. A general objective of the proposed research is the development of new genetic methods that can be widely applied to the study of biologically important protein-protein interactions. A recently developed E. coli- based two-hybrid assay will be tested for its efficacy in detecting interacting proteins encoded in complex libraries. This assay will also be applied to the study and manipulation of a phosphorylation-dependent protein-protein interaction involving the eukaryotic transcription factor CREB and its CREB and its co-activator CBP.