Interaction of two classes of release factors from Euplotes octocarinatus.
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 x 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.
Chai
Bao-Feng
BF
Key Laboratory of Chemical Biology and Molecular Engineering of Education Ministry, Shanxi University, Taiyuan 030006, China. bfchai@sxu.edu.cn
Song
Li
L
Fu
Yue-Jun
YJ
Wang
Wei
W
Liang
Ai-Hua
AH
D000818:Animals; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D016812:Euplotes / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010454:Peptide Termination Factors / Q000235:genetics / Q000502:physiology*; D010802:Phylogeny; D010957:Plasmids
D003062:Codon; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; C087971:peptide-chain-release factor 3; C489005:prfA protein, E coli; C489006:prfB protein, E coli; C489007:prfC protein, E coli