PROTEIN/PROTEIN INTERACTIONS IN PROKARYOTIC GENE CONTROL

dna结合调节蛋白与RNA聚合酶（或相关因子）相互作用，在转录水平上控制基因表达。拟议研究的长期目标是更好地了解dna结合调节剂如何激活和抑制原核生物的转录。原核生物调控系统的相对简单性促进了基础机制的研究，为研究高等生物的转录调控提供了基础。RNA聚合酶（RNAP）与许多天然激活剂的相互作用已被广泛研究。与RNAP融合的异源蛋白结构域可以通过为dna结合蛋白提供接触面来介导转录激活，这为探索转录激活机制提供了一个有价值的新工具。这种蛋白质-蛋白质接触激活转录的机制将通过体外和体内实验进行检验。迄今为止研究的大多数原核激活剂似乎与RNAP的α或sigma亚基相互作用。与sigma相互作用的一种特别具有良好特征的激活剂是噬菌体λ cI蛋白。我们将研究sigma亚基在转录激活中的作用，并验证lambdacI通过稳定sigma域与启动子-35区域的结合来激活转录的假设。在原核生物中，转录抑制的机制是与RNAP的简单竞争性结合。然而，最近的证据表明，一些抑制因子可以通过与RNAP接触来发挥作用。dna结合蛋白与RNAP之间的接触足以抑制和激活转录的假设将得到验证。

DNA-bound regulatory proteins interact with RNA polymerase (or associated factors) to control gene expression at the transcriptional level. The long-term goal of the proposed research is to better understand how DNA-bound regulators activate and repress transcription in prokaryotes. The relative simplicity of prokaryotic regulatory systems facilitates basic mechanistic studies that can provide a foundation for the study of transcriptional regulation in higher organisms. The interactions of RNA polymerase (RNAP) with a number of natural activators have been extensively studied. The demonstration that heterologous protein domains fused to RNAP can mediate transcriptional activation simply by providing contact surfaces for DNA-bound proteins provides a valuable new tool with which to explore the mechanism of transcriptional activation. The mechanisms by which such protein-protein contacts activate transcription will be examined by experiments performed in vitro and in vivo. Most prokaryotic activators that have so far been examined appear to interact with either the alpha or the sigma subunit of RNAP. A particularly well-characterized activator that interacts with sigma is the bacteriophage lambda cI protein. The role of the sigma subunit in transcriptional activation will be examined, and the hypothesis that lambdacI activates transcription by stabilizing the binding of a domain of sigma to the promoter -35 region will be tested. A well-established mechanism for transcriptional repression in prokaryotes is simple competitive binding with RNAP. However, recent evidence suggests that some repressors can exert their effects through contact with RNAP. The hypothesis that contact between a DNA-bound protein and RNAP can suffice to repress as well as activate transcription will be tested.