| In eukaryotic protein translation process,when one of the three codons,UAA,UAG or UGA,enters the ribosomal A site,is recognized by eukaryotic polypeptide release factor(eRF1),translation termination occurs.Cooperated with the eukaryotic polypeptide release factor(eRF3),complting the nascent peptide release and the cycle of the ribosome.Translation termination is performed by eRF1,which recognizes stop codons via its N-terminal domain.Conserved motifs,such as GTS,NIKS and Y×C×××F in N-domain of eRF1,were found to be important for stop codon recognition.However,the detailed decoding mechanism,especially the selective recognition of stop codon remains unclear.Firstly,in our experiment we constructed chimerics Tt/Sc eRF1 and Tt/Sp eRF1 that contained the eRF1 N domain of Tetrahymena thermophilia and the M,C domain of Saccharomyces cerevisiae or Schizosaccharomyces pombe.Using the dual-luciferase read-through report system in chimerics,confirmed that two kinds of the hybrid e RF1 in recognition of the stop codon activity had significant difference.We found that Tt/Sc eRF1 recognized stop codon UGA,while the UAG and UAA of read-through efficiency is significantly higher than the UGA,similared to the eRF1 of Tetrahymena,showed the stop codon recognition specificity(Read-through level at UGA 1.98%,UAG 3.83%,UAA 11.04%,respectively).However,Tt/Sp eRF1 can recognize three stop codons,no recognized specificity(Read-through level at UGA 2.81%,UAG 2.01%,UAA1.88%,respectively).To illustrate that the N-domain of Tt-eRF1 is not the only factor of recognition specilicity.In the M,C domain of eRF1 may be exist other componets or factors in the cell,or these factors may be participate in recognizing stop codon of eRF.To explain this phenomenon,the C-terminal minidomain of Schizosaccharomyces pombe eRF1 of the key amino acids mutated,choose one of the 357,361,364 and 367,acquired that a series ofmutants(Tt/Sp eRF1 A364 Q,Tt/Sp eRF1 D361A/A364 Q,Tt/Sp eRF1L357A/D361A/A364 Q,Tt/Sp eRF1L357A/D361A/A364Q/E371S).Similarly,chose the 364,367 and 371 amino acids of Schizosaccharomyces pombe,were also introduced into Saccharomyces cerevisiae,and obtained mutants(Tt/Sc e RF1 Q364 A,Tt/Sc eRF1 Q364A/D367 E,Tt/Sc eRF1 Q364A/D367E/E371S).The recults show that all of the mutants had no obvious change about the UGA recognition.but the readthrough efficiency of Tt/Sp eRF1 mutants about the UAG and UAA increased significantly,indicating that the recognition efficiency of the two codons reduced.Meanwhile,Tt/Sc eRF1 mutants,no significant influence on the recognition activity of all the stop codons,but the recognition activity of UAG increased slightly,the percent read-through from 5.77% educed to2.98%.The above results that the C-terminal Minidomain affect the recognition activity of UAG and UAA.In order to futher analyze the influence of the minidomain,analyzing the protein structure of the minidomain,which found a key,changeful Loop(357~367aa),so we use the C-terminal minidomain Loop of Tetrahymena thermophilia and Saccharomyces cerevisiae Loop introduced to the hybrid Tt/Sc eRF1.The results that the two different Loop Tt/Sc e RF1 can recognize three stop codons,indicating that the Loop structure had a significant influence on the recognition activity.The results showed that the recoginiton activity about N-terminal domain of Tetrahymena thermophilia eRF1 was affected by the N-terminal domain,which indicated that C-terminal domain of eRF1 was involved in the process of stop codon decoding.From the above experimental results,we speculate that,whether the interaction efficiency between the Sc eRF3 and the hybrid eRF1 s is different,due to the change of the two chimeras recognition efficiency and function.Therefor,in this study,we cloned the releases factors,the hybrid eRF1 of Tetrahymena eRF1,S.pombe eRF1 and the two classes of releases factors Sc-eRF1 and Sc-eRF3.After subcloning them into the vector pGADT7 and pGBKT7,respectively,we transformed the recombinantplasmids into the yeast strain AH109 to assay interaction between two classes of release factor.The yeast two hybrid technology confirmed that the Tetrahymena e RF1 chimeras(Tt/Sc eRF1/Tt/Sp eRF1),S.pombe eRF1,Sc-eRF1 and Sc-eRF3 could interact in vivo,suggest that no matter in which kind of cells,protein translation termination process requires two classes of release factor to cooperated to complete.And exclude that the difference of the activity of the recognition of hybrid eRF1 s,which found to be due to the different effiency of the interaction between the Sc eRF3 and the hybrid eRF1 s.In order to confirmed that the N-terminal domain recognition activity affected by the C-terminal domain,we use the Bimolecular fluorescence complementation system(BiFC)confirmed the main regulated factors of eRF1 functional is caused by interaction of the C-terminal domain an N-terminal domain.We cloned the N-terminal domain,C-terminal domain of Tetrahymena eRF1,the C-terminal domain of S.cerevisiae,the C-terminal domain of S.pombe and its mutants(Sp-C A364Q/ Sp-C D364A/A364 Q /Sp-C L357A/D364A/A364Q).After subcloning them into the vector pQE-30 and pET-28 a,respectively.The results showed that the N-terminal domain of Tt eRF1 and the different C-terminal domain,mutants can interact in vivo,indicating that the eRF1 recognize stop codom specificity depends on intramolecular interaction between the domain of eRF1.Also the C-terminal domain of eRF1 modulates the process of stop codon decoing.Using Bimolecular fluorescence complementation system to confirm the N-terminal domain and C-terminal domain action in vivo,suggesting that Tetrahymena eRF1 recognize stop codom specificity depends on intramolecular interaction between the domain of eRF1.Comprehensive the above results shows that the N-terminal domain of Tetrahymena eRF1 is not the only factor about recognize the stop codon,C-terminal domain influences and determine the eRF1 specificity for stop codons to a certain extent.As a result,this study provide clues for elucidating the mechanism of stop codon recognition by polypeptide release factors. |
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