【作者】 王伟； 智慧； 张志云； 柴宝峰； 梁爱华；

【Author】 WANG Wei, ZHI Hui, ZHANG Zhi-Yun, CHAI Bao-Feng, LIANG Ai-Hua Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006, China

【机构】 山西大学生物技术研究所化学生物与分子工程教育部重点实验室；

【摘要】 细菌中蛋白质合成的终止需要两类肽链释放因子,第一类释放因子RF1/RF2和第二类释放因子RF3。两类释放因子的相互作用促使肽链合成的终止和新生肽链的释放。本研究将全长和截短型的赭纤虫释放因子1 表达质粒pRSETc—BjeRF1、pRSETc—BjeRF1(1—270)、pRSETc-EoRab以及表达载体pRSETc,分别转化大肠杆菌细胞BL21(DE3),PCR鉴定,证明获得含有重组不同表达质粒的大肠杆菌BL21(DE3)细胞克隆。不同的克隆在37℃的LB液体培养基中生长,SDS-PAGE和Western-blotting检测,表明全长和截短型的赭纤虫释放因子1以及EoRab异源蛋白在宿主细胞中都有表达。对含有不同质粒转化子的大肠杆菌的生长通过光密度法跟踪检测。发现全长赭纤虫释放因子的本底表达显著抑制了大肠杆菌细胞在对数生长期的增殖。分析表明,赭纤虫释放因子1和大肠杆菌的释放因子3在细胞中很可能发生了相互作用,由此影响了大肠杆菌释放因子3与核糖体上大肠杆菌释放因子1的作用,释放因子1在核糖体上的循环受阻,蛋白质合成减缓,细胞生长抑制更多还原

【Abstract】 The termination of protein synthesis in bacteria requires two codon-specific polypeptide release factors, RF-1 and RF-2. A third factor, RF-3, which accelerate the dissociation of RF1 and RF2 from ribosomes after hydrolysis of peptidyl-tRNA. In this study, expression plasmid pRSETc-BjeRF1, pRSETc-BjeRFl (1-270), pRSETc-EoRab and expression vector pRSETc were transformed into E.coli cell respectively. Different transformants were grown at 37℃ in LB medium. Expression of exogenously protein BjeRF1, BjeRFl (1-270) and EoRab in E. coli BL21 (DE3) was analyzed by 12.5% SDS-PAGE. Western-blotting assay with anti-His antibody further confirmed that expression of exogenously protein BjeRF1, BjeRFl (1-270) and EoRab in E.coli BL21 (DE3). Compared to the control BjeRF1 (1-270) and EoRab, expression of BjeRFl affected E.coli cell growth significantly. The results suggested that BjeRFl interact probably directly with RF3. The interaction between BjeRFl and RF3 affected interaction between RF1 / RF2 and RF3, the recycling of RF1 and RF2 is inhibited between different ribosome. Accurate translation termination is affected and the cell growth is inhibited. Our data support the prediction that RF1/RF2 and eRF1 may have comparable structures on their respective ribosomes and termination is likely to be based on similar mechanisms across species更多还原