CONTROL OF TRANSCRIPTION TERMINATION IN E COLI

对温带噬菌体的研究在产生关于普遍调节机制的信息方面非常富有成果。通过抑制或诱导转录终止来控制基因表达，最初在噬菌体中被描述，现在已知在真核细胞中很重要，特别是在控制c-myc和HIV基因表达方面。在lambda中，N蛋白的抗终止允许噬菌体生长，而由竞争噬菌体的Nun蛋白诱导的终止，HK022阻断lambda的发育。109 aa Nun蛋白是富含精氨酸的RNA结合蛋白ARM家族的成员，该家族包括N蛋白、HIV-1 Tat蛋白和HIV-1 Rev蛋白。本文拟从生物化学和遗传学两方面研究其作用机制。通过核磁共振、荧光各向异性和交联策略来探测Nun配合物与RNA、宿主NusA因子或RNA聚合酶的结构。研究人员正在探索Nun的c端与DNA模板之间可能的相互作用。Nun的c端携带一个新的Zn2+结合基序，这是终止所必需的；该配体在非促进反应中的作用有待研究。化学研究将与尼姑和宿主蛋白的诱变一起进行，以确定相互作用的残基。在Nun终止和细胞杀伤中起作用的宿主因子将被定义为遗传学和生物化学特征。其中包括NusG，一种被认为释放Nun-阻滞转录延伸复合物的因子，以及一种允许被Nun蛋白杀死宿主的因子。

The study of the temperate bacteriophage has been exceedingly fruitful in generating information about universal regulatory mechanisms. Control of gene expression by suppression or induction of transcription termination, first described in phage, is now known to be important in eukaryotic cells, in particular in the control of c-myc and of HIV gene expression. In lambda, antitermination by the N protein allows phage growth, whereas termination induced by the Nun protein of the rival phage, HK022 blocks lambda development. The 109 aa Nun protein is a member of the arginine-rich RNA binding protein ARM family that includes N, HIV-1 Tat, and HIV-1 Rev proteins. This proposal is to study the mechanism of action of Nun through biochemical and genetic approaches. The structures of Nun complexes with RNA, the host NusA factor, or RNA polymerase will be probed by NMR, fluorescence ainsotropy and crosslinking strategies. A possible interaction between the C-terminus of Nun and the DNA template is to be explored. The C-terminus of Nun carries a novel Zn2+ binding motif that is essential for termination; the role of this ligand in Nun-promoted reactions is to be studied. Chemical studies will be performed in concert with mutagenesis of Nun and host proteins to define interacting residues. Host factors that play a role in Nun termination and cell killing are to be defined genetically and characterized b2iochemically. These include NusG, a putative factor that releases Nun-arrested transcription elongation complexes, and a factor that allows host killing by the Nun protein.