Structure-function relationships of transcription termination factor Rho

大肠杆菌转录终止因子rho是46 kDa亚基的六聚体，催化从三元转录复合物中释放新生RNA。已知rho单体具有三个主要结构域：N-末端三分之一具有RNA结合活性，中心250-aa区域携带ATP结合位点和C-末端未知功能的剩余部分。为了进一步阐明rho蛋白的精确结构-功能关系，我们使用改进的PCR反应将随机突变引入其单个结构域。将诱变的rho等位基因克隆到乳糖启动子控制的表达载体pEC 22中，并选择其受损的rho依赖性终止活性。我们首先集中在特征最少的C-末端结构域，并分离出53个在rho编码序列的3 '-末端310 nt区域内携带点突变的克隆。到目前为止，rho蛋白从两个这样的突变体，E342 G和A357 V，纯化和体外表征。结果表明，这两个突变都影响了rho蛋白对RNA的识别方式和ATP的水解效率。因此，C端结构域在调节和协调RNA结合结构域和ATP结合结构域的功能方面具有重要作用。

The Escherichia coli transcription termination factor rho is a hexamer of 46kDa subunits and catalyzes the release of the nascent RNA from the ternary transcription complex. A rho monomer is known to have three major structural domains : the N-terminal one third possessing an RNA binding activity, the central 250-aa region carrying the ATP binding site and the C-terminal remainder of an unknown function (s). To further elucidate the precise structure-function relationships of rho protein, we introduced random mutations into its individual structural domains using a modified PCR reaction. The mutagenized rho alleles were cloned into a lac promoter-controlled expression vector , pEC22, and selected for their impaired rho-dependent termination activities. We first focused on the least-characterized C-terminal domain, and isolated 53 clones carrying point mutations within the 3'-terminal 310nt region of the rho-coding sequence. Thus far, rho proteins from two such mutants, E342G and A357V, were purified and characterized in vitro. The results revealed that the both mutations affect the mode of recognition of RNA and the efficiency of ATP hydrolysis by rho protein Thus the C-terminal domain must have a vital role in regulating and coordinating the functions of the RNA binding and ATP binding domains.