八肋游仆虫eRF1a的C端是肽链释放因子eRF3的结合区(英文)

真核生物蛋白质合成终止需要两类肽链释放因子,eRF1和eRF3.研究表明八肋游仆虫的两类肽链释放因子在体内和体外都能形成复合物,且第一类肽链释放因子eRF1a与第二类肽链释放因子eRF3的C端结合.为了确定eRF3在eRF1a上的结合区,本研究以八肋游仆虫第一类肽链释放因子eRF1a基因为模板,用PCR的方法获得了N端分别截短140和206个氨基酸的eRF1a片段,同时在这两个片段的3'端融合了编码6个组氨酸残基的核苷酸序列,将这两个序列分别插入原核表达载体pTWIN1,并构建重组表达质粒pTWIN1-eRF1aC1 his6和pTWIN1-eRF1 aC2his6,转入大肠杆菌BL21( DE3)中获得了可溶性表达,通过一步His60 Ni Superflow柱亲和层析,重组蛋白CBD-intein-eRF1 aC1 his6和CBD-intein-eRF1aC2 his6获得纯化.体外pull down分析显示eRF1aC1和eRF1aC2均能与八肋游仆虫第二类释放因子eRF3相互作用,这表明八肋游仆虫eRF1a的C端是肽链释放因子eRF3的结合区.

C Terminus of eRF1a is the Binding Region of Release Factor eRF3 in Euplotes octocarinatus

In eukaryotes,translation termination is mediated by two polypeptide chain-release factors,eRF1and eRF3.In Euplotes octocarinatus,eRF1and eRF3forms complex in vivo and in vitro,and the eRF1abinding region on eRF3 are located at the C terminus of eRF3.To identify the eRF3binding region on eRF1a,the gene-coding regions of two N-terminal truncated eRF1a,eRF1aC1(aa 141-447) and eRF1aC2(aa 207-447) were inserted into pTWIN1vector fused in frame with an DnaB intein gene;meanwhile,a hexahistidine sequence was fused to the C terminus of CBD-intein-eRF1aC1and CBD-intein-eRF1aC2.The recombinant plasmids pTWIN1-eRF1aC1his6and pTWIN1eRF1aC2his 6 were transformed into the strain E.coli BL21(DE3).The recombinant CBD-intein-eRF1aC1his 6 and CBD-intein-eRF1aC2his 6 proteins were expressed and then purified by one-step affinity chromatography using His60 Ni Superflow column.In vitro pull-down analysis showed that the recombinant CBD-intein-eRF1aC1his 6 and CBD-intein-eRF1aC2his 6 interacted with E.octocarinatus polypeptide chain release factor eRF3.This result suggested that C terminus of eRF1awere the binding region of release factor eRF3in Euplotes octocarinatus.