Ghosh B, Das A
Proc Natl Acad Sci U S A. 1984 Oct;81(20):6305-9. doi: 10.1073/pnas.81.20.6305.
We demonstrate that the protein product of the Escherichia coli nusB gene is essential for transcription antitermination in vitro by phage lambda N gene product. We recently have described a convenient biochemical assay for N protein activity in the S30-coupled transcription translation system and demonstrated that N action requires the 69-kDa L factor (nusA), the product of E. coli nusA gene. Using a complementation assay for the restoration of N activity specifically in the nusB mutant extract, we have purified the nusB complementing activity. This activity is due to a 15-kDa polypeptide that is overproduced in E. coli containing multiple copies of the nusB gene. We find that nusA and nusB are required for N activity to suppress a rho-dependent as well as a rho-independent terminator. The requirement for nusB protein in antitermination could not be overcome by an excess of nusA or N protein, nor could an excess of nusB overcome the requirements for nusA in antitermination. Our results suggest that the formation of an antitermination apparatus by N requires nusA and nusB proteins in equimolar amounts.
我们证明，大肠杆菌nusB基因的蛋白质产物对于噬菌体λ N基因产物在体外的转录抗终止作用至关重要。我们最近描述了一种在S30偶联转录翻译系统中检测N蛋白活性的简便生化方法，并证明N的作用需要69 kDa的L因子（nusA），即大肠杆菌nusA基因的产物。通过在nusB突变体提取物中特异性恢复N活性的互补试验，我们纯化了nusB互补活性。这种活性归因于一种15 kDa的多肽，它在含有多个nusB基因拷贝的大肠杆菌中过量产生。我们发现，nusA和nusB是N活性抑制ρ依赖性和ρ非依赖性终止子所必需的。过量的nusA或N蛋白无法克服抗终止对nusB蛋白的需求，过量的nusB也无法克服抗终止对nusA的需求。我们的结果表明，N形成抗终止装置需要等摩尔量的nusA和nusB蛋白。
