八肋游仆虫两类释放因子的相互作用(英文)

Interaction of Two Classes of Release Factors from Euplotes octocarinatus

从八肋游仆虫中克隆到两类释放因子基因Eo eRF1和Eo eRF3。在Eo eRF3基因的阅读框中有 3个通用的终止密码子UGA ,在此编码半胱氨酸。为了研究两类释放因子的相互作用 ,用PCR的方法对 3个位点进行了定点突变 ,将UGA突变为通用的编码半胱氨酸的密码子UGU。突变结果经测序确认后 ,在大肠杆菌中获得全长Eo eRF3的正确表达。在此基础上 ,构建酵母双杂交重组质粒 ,用该系统检测了游仆虫两类释放因子的相互作用。结果显示 ,两类释放因子在生物体内形成复合体 ,从而在较原始的真核生物中 ,证实了两类释放因子的相互作用关系。

Translation termination on the ribosome is an essential process for cell viability.This process is maintained by two classes of peptide release factors (RF1/RF2,RF3 and eRF1,eRF3 in prokaryotes and eukaryotes,respectively).In protozoa ciliates Euplotes octocarinatus,an unicellular eukaryotes,universal stop codon UGA is reassigned for cysteine suggesting the specificity of evolution of translation termination system.We cloned two classes of release factors from Euplotes octocarinatus previously.In this paper,three in-frame stop codons UGA in Eo-eRF3 gene were mutated mediated by PCR site directed mutagenesis method.The interaction between eRF1 and eRF3 from E.octocarinatus was assayed in vivo using Yeast Two-hybrid System,which has an advantage of highly sensitivity.The results showed that the eRF1·eRF3 complex was formed in living cells to function in the process of translation termination,differing from that in prokaryotes in which (RF1/RF2) and RF3 function separately.The evolution of translation termination of life-form was analyzed using phylogenetic tree of amino acids sequences of RFs (32(e)RF1s and 24(e)RF3s) obtained from GenBank.Two classes of RFs are useful information in analysis of evolution of life-form and further elucidation of mechanism of translation termination of protein synthesis on ribosome.