| Translation is one of important bioprocesses in cells, which involves the interactions between or among RNA and proteins. This process includes initiation, elongation and termination. Former two are regulated by initiation factors and elongation factors, repectively; and the polypeptide release factors are reponsible for the stop codon recognition and nascent polypiptide release from ribosome.Class I release factor in eukaryotes is highly conseved in both primary and tertiary structure, which contains three functional domains named N, M and C domain. Domain N is putatively responsible for stop codon recognition in A site of ribosome, which contains two conseved motifs, "TASNIKS" and "Y×C×××F"; M domain is responsible for the hydrolysis of easter bond of peptidyl-tRNA leading to the release of nascent polypeptides; C domain is responsible for interaction between eRF1 and eRF3 which is an eRF1 and ribosome dependent GTPase.Class I release factors in high eukaryotes can recognize all three stop codons to accomplish the termination process of translation. The stop codons are reassigned in ciliates of protozoan, such as UAA and UAG encode Glu, while UGA is used as stop codon in Paramecium, Tetrahymena and Stylonychia; however, in Euplotes, universal stop codon UGA encode Cys, while UAA and UAG are used as stop codons. These phenomena suggest us the evolutionary of characteristics and structure of polypeptide release factors. Giardia lamblia is a primary organism in protozoan, which potentially formed before the formation of mitochondrion. The structure of classⅡrelease factor of G. lamblia is significant different from that of high eukaryotes, even that of ciliates. This would provide us an ideal model for probing the evolutionary mechanism of termination of protein synthesis.Two classes of release factor gene, GeRF1 and GeRF3 have been cloned from the genomic DNA of G. lamblia. Both of them were subcloned into the plasmid pGADT7 and pGBKT7, respectively, and then transformed into the yeast strain AH 109 for interaction assay. The results indicated that GeRF1 and GeRF3 could interact in vivo, suggesting that both of release factors cooperatively accomplish the termination process of protein synthesis in G. lamblia.Many studies demonstrated that N domain of eRF1 was responsible for stop codon recognition, so we constructed a hybride gene Gl/Sc eRFl containing N domain of G. lamblia GeRF1 and M and C domains of Saccharomyces cerevisea eRF1 to produce a fused protein Gl/Sc eRF1 in yeast cell. The sup45 (eRF1) knockout yeast strain YDB447 was used to assay the function and characteristics of the Gl/Sc eRF1. The hybrid gene Gl/Sc eRF1 was subcloned into plasmid pDB0984, then transformed into YDB447 containing wild type sup45 (eRF1) in plasmid pUKC802, which can be evicted by pressure of 5'-FOA in solid SD medium. Viability of yeast cells were determined by activity of Gl/Sc eRF1 as a sole polypeptide release factor. The results demonstrated that Gl/Sc eRF1 could not recognize three stop codons meantime.Further assays of the activity and charaterization of Gl/Sc eRF1 were conducted by using dual-luciferase report system. The results confirmed that Gl/Sc eRF1 could not recognize either of stop codons. Mutagenesis (T26L, G48K, N112E T113N, S134N) were performed to introduce the amino ancids into Gl/Sc eRF1, which were same to that in high orgnisms. These mutants also could not significant change the acvtivity of stop codons discrimination of Gl/Sc eRF1.This paper demonstrated that the N terminal domain of eRF1 is not the sole determinant for stop codon discrimination in G. lamblia. Other factors, such as eRF3, and determinants, such as M and/or C domian may participate in regulation of stop codon recognition of eRF1.
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