Expression And Function Analysis Of Eukaryotic Polypeptide Release Factor

In protein synthesis process,when one of the UAA,UAG and UGA reaches the ribosomal A site,eRF1 recognizes the stop codon,and works together with eRF3 to complete release of the nascent peptide chain.Ciliates’ eRF1 displays obvious specificity of stop codon recognition.It is possible that the changes of some key amino acids or motifs cause eRF1 losing the ability to recognize one of the three bases of stop codons.Identification of these residues or motifs will help to clarify the molecular mechanism of the recognition of the stop codon by eRF1.Currently,the crystal structure of prokaryotes RF1 and RF2,and human eRF1 have been obtained.It promoted the progress of functional studies of eRF1,and various recognition models were established based on it.Researches of the specificity of ciliates’ stop codon recognition are mainly based on the structure of human eRP1.What are the differences in the structure between the eRF1s of human and ciliates?There is currently no data in this respect,so it is necessary to carry out analysis and comparative study of ciliates’ eRF1 structure to reveal the molecular mechanism of the stop codon recognition in different evolutionary statuses.eRF3 is a GTPase interacting with eRF1 to promote the release activity of eRP1.In addition,eRF3 also has a variety of functions in cells,such as cell cycle regulation,cytoskeleton and cell apoptosis etc.Survivin is the smallest member of the inhibitor of apoptosis(IAP)proteins family.It can not only inhibit apoptosis,but also promote cell transformation and involve in cell mitosis,angiogenesis and the drug resistance of tumor cells.Both eRF3 and survivin have the related function in the regulation of cell cycle,apoptosis and tumorigenesis,therefore,the interaction relationship between them is a question being worth exploring.In the previous studies,we have demonstrated for the first time that the human eRF3 and survivin can recognize and interact with each other in vitro and vivo,and we obtained the deletion mutants of eRF3a,eRF3b and survivin,thereby having determined the primary interaction area of eRF3 and survivin.In this study,we further truncated eRF3a,and successively verified the interaction relationship between eRF3a(1-72aa)/eRF3a(1-36aa)and survivin by yeast two-hybrid technique and pull-down analysis identified by Western blot technique.The results show that both eRF3a(1-72aa)and eRF3a(1-36aa)can recognize and interact with survivin in vivo and vitro,therefore,we confirmed the smaller interacting domain of eRF3 between the 1 and 36 amino acids and provided a data support for the further confirming of eRF3 cooperating with survivin involving in cell cycle and apoptosis regulation.To accurately answer this question,we need the structure data of the peptide chain release factor.We have obtained some data by the research of human peptide chain release factor and eRF1 of ciliates,such as Euplotes and Tetrahymena,and found some motifs and amino acid residues related to the stop codon recognition.To further find out the dif-ferences reflected in the structures of eRF1,we expressed the full-length Euplotes eRF1a(Eo-eRF1a)and eRF1b(Eo-eRF1b),Giardia eRF1(G1-eRF1)and eRF3 and Trichomonas eRF3 in Pichia pastoris.In order to obtain the efficiently expressed and correctly modified eRF1,we optimized gene code of Euplotes eRF1 a and synthesis the full-length gene.Then we cloned these eRF1 and eRF3 into Pichia pastoris expression vector pPIC9 and pPICZaA,respectively.After linearized with Bgl II or Sac I,eRF1 and eRF3(with promoter and secret leading peptide)were transformed into Pichia pastoris GS115 strain with LiCl transformation method.The screened recombinants were induced with methanol.However,the full-length proteins of polypeptide release factors have not been efficiently expressed though vary conditions were considerated.Numerous studies showed that the N-terminal domain of eRF1 determined the function of stop codons recognition by eRF1.For obtaining structural data,we constructed a series of prokaryotic recombinant expression plasmid containing gene fragments of N-terminal domain,Eo-eRF1b-N,Tt-eRF1-N and G1-eRF1-N.They were soluble expressed in E.coli,and the target proteins were identified with Western blotting.This will provide us supports for next research on the structure of polypeptide release factors.