A Genetic Approach to Mapping Activation Target Surfaces on E. coli RNA Polymerase

已知原核生物中的许多转录激活因子在启动子附近结合并接触RNA聚合酶（RNAP）。然而，对天然激活剂的研究尚未确定dna结合激活剂与RNAP之间的蛋白-蛋白接触是否足以激活转录。最近在首席研究员实验室的工作表明，dna结合蛋白与融合到RNAP的异源蛋白结构域之间的接触可以引发转录激活，这表明dna结合蛋白与RNAP的任何可接近表面之间的接触都会激活转录。本研究的一个主要目的是批判性地评估这一假设，并生成大肠杆菌RNAP表面的遗传图谱，这些表面可能被dna结合激活蛋白所接近。为此，将采用一种遗传方法来筛选那些当与位于启动子上游的dna结合蛋白融合时激活转录的随机肽。遗传和互补生化方法将用于确定这些肽接触的RNAP上的位点。除了定义RNAP的可接近表面外，这些人工肽激活剂本身可能被证明是有用的生物试剂。具体来说，它们将被用于产生一组体内抑制剂，这些抑制剂可能有助于鉴定大肠杆菌中天然活化剂的激活靶点。这些研究将提供大肠杆菌RNAP表面转录激活剂的观点。除了为RNAP与控制其功能的调控因子的相互作用提供新的见解外，这些研究的结果应该与蛋白质-蛋白质和肽-蛋白质相互作用的研究普遍相关。

MCB9728765 Hochschild Many transcriptional activators in prokaryotes are known to bind near a promoter and contact RNA polymerase (RNAP). The study of natural activators has not, however, established whether a protein-protein contact between a DNA-bound activator and RNAP suffices to activate transcription. Recent work in the Principal Investigator's lab has shown that contact between a DNA-bound protein and a heterologous protein domain fused to RNAP can elicit transcriptional activation, suggesting that contact between a DNA-bound protein and any accessible surface of RNAP will activate transcription. A main objective of this research is to critically evaluate this hypothesis and to generate a genetic map of the surfaces of E. coli RNAP that are potentially accessible to DNA-bound activator proteins. To this end, a genetic method will be employed to screen random peptides for those that activate transcription when fused to a DNA-bound protein positioned upstream of a promoter. Genetic and complementary biochemical methods will be used to identify the sites on RNAP that are contacted by these peptides. As well as defining the accessible surfaces of RNAP, these artificial peptide activators may prove useful as biological reagents in their own right. Specifically, they will be used to generate a panel of in vivo inhibitors that may facilitate the identification of activation target sites for natural activators in E. coli. These studies should provide a view of the surface of E. coli RNAP as seen by transcriptional activators. In addition to offering new insight into the interactions of RNAP with regulators that control its function, the results of these investigations should be generally relevant to the study of protein-protein and peptide-protein interactions.