GENE SELECTION BY MULTIPLE EUKARYOTIC RNA POLYMERASES

这是一个研究rRNA转录机制及其在植物中的应用的建议。 真核生物中的调节。 一种体外转录系统， 克隆的rDNA、均相RNA聚合酶I（RNAP I）和辅助的 蛋白质（转录起始因子-TIF）被用于：（1） 确定TIF的数量，并将其纯化至均匀，（2） 更详细地确定TIF在启动 转录，（3）以确定TIF和RNAP I如何相互作用， 其他和模板和（4）描述动力学的事件 参与起始复合物的组装。 DNA足迹和 化学改性保护实验将用于确定 蛋白质-DNA接触。 化学交联实验将用于 确定蛋白质-蛋白质相互作用。 将使用动力学测量 以确定引发的动力学机制。 单克隆抗体 RNAP I亚单位和TIF将被生产，以帮助 分析. rRNA基因表达的转录是发育性的 通过RNAP I的修饰在阿米巴中调节。 实验来 鉴定修饰的化学性质和催化的酶 建议。 在启蒙过程中的哪一步 受修改的影响也将利用技术进行研究 建议研究引发机理。

This is a proposal to study the mechanism of rRNA transcription and its regulation in eukaryotes. An in vitro transcription system made up of cloned rDNA, homogeneous RNA polymerase I (RNAP I) and an auxilliary protein (transcription initiation factor(s)-TIF) is being utilized: (1) to determine the number of TIFs, and to purify them to homogeneity, (2) to determine in greater detail the role played by the TIF in initiation of transcription, (3) to determine how the TIF and RNAP I interact with each other and with the template and (4) to describe kinetically the events involved in the assembly of the initiation complex. DNA footprinting and chemical modification-protection experiments will be used to determine protein-DNA contacts. Chemical cross-linking experiments will be used to determine protein-protein interactions. Kinetic measurements will be used to determine the kinetic mechanism of initiation. Monoclonal antibodies to the RNAP I subunits and the TIF(s) will be produced to aid in the analysis. Transcription of rRNA gene expression is developmentally regulated in Acanthamoeba by modification of RNAP I. Experiments to identify the chemical nature of the modification and the enzyme catalyzing it are proposed. Exactly which step in the process of initiation is affected by the modification is also to be studied utilizing the techniques proposed to study the mechanism of initiation.