GENE REGULATION BY TRANSCRIPTION ANTITERMINATION

该提案的主要目标是阐明作用机制 一种典型的抗终止剂，由温和噬菌体编码的N蛋白 阿卡德 N是一种小的模块化蛋白质， 噬菌体基因组上的遗传信号（称为nut）和一些宿主 蛋白质（称为Nus因子）修饰宿主RNA聚合酶， 抗终止模式。 目前的工作模式建议， 存在于N中的精氨酸基序结合由nut编码的boxB RNA发夹， 使N与靶聚合酶紧密接触。 N然后捕获 聚合酶通过mRNA环和衔接子宿主因子的协作 （NusA），并通过作为一个终止子引导RNA聚合酶通过下游终止子。 操纵子特异性亚基 其他宿主因子与保守的 RNA信号和N，以促进组装以及持久 抗终止 拟议中的实验将联合收割机结合遗传， 生物化学和生物物理方法来完成几个主要的 目标. 第一个目标是执行几条机械路线 研究严格测试模型的各种预测，进一步 剖析核心成分在终止抑制中的作用， 阐明了潜在的分子机制。 第二个目标是 通过独立遗传学方法分离缺失的宿主因子及其基因， 和生物化学方法，并阐明其在组装的功能， N-抗终止复合物，持久抗终止，和在转换 噬菌体从溶源性发展到裂解模式。 第三个目标是 用分子遗传学和生物物理学方法研究N-boxB相互作用 来破译控制RNA-蛋白质相互作用的化学原理。 第四个目标，该项目的一个新方向，是识别细胞 通过N样抗终止机制激活的基因，并分离 N. 这些研究将提供 关于结构、功能和调节的基本信息， 原核生物的转录器，应该阐明RNA的功能 信号在转录控制，并应揭示的方面， RNA信号序列特异性识别的分子基础 调节蛋白

The primary goal of this proposal is to elucidate the mechanism of action of a model antiterminator, the N protein encoded by temperate phage lambdal. N is a small, modular protein that interacts with a specific genetic signal (called nut) on the phage genome and a number of host proteins (called Nus factors) to modify host RNA polymerase to a termination-resistant mode. The current working model proposes that an arginine motif present in N binds the boxB RNA hairpin encoded by nut, bringing N in close contact with the target polymerase. N then captures polymerase through mRNA looping and collaboration of an adapter host factor (NusA), and leads RNA polymerase through downstream terminators by being an operon-specific subunit. Additional host factors interact with conserved RNA signals and N to facilitate both the assembly as well as persistent antitermination. The proposed experiments will combine genetic, biochemical and biophysical approaches to accomplish several major objectives. The first goal is to carry out several lines of mechanistic studies to rigorously test various predictions of the model, further dissecting the role of the core components in termination-suppression and elucidating the underlying molecular mechanisms. The second goal is to isolate the missing host factor(s) and their genes by independent genetic and biochemical approaches and elucidate their function in assembly of the N-antitermination complex, persistent antitermination, and in switching phage development from lysogenic to the lytic mode. The third goal is to study N-boxB interaction with molecular genetic and biophysical approaches to decipher the chemical principles that govern RNA-protein interaction. The fourth goal, a new direction of the project, is to identify cellular genes that are activated by N-like antitermination mechanism, and isolate potential cellular homologues of N. These studies should provide fundamental information on the structure, function and regulation of the prokaryotic transcription apparatus, should illuminate the function of RNA signals in transcription control, and should reveal aspects of the molecular basis of sequence-specific recognition of RNA signals by regulatory proteins.