Study On The Functional Domains Of Ciliates ERF1 Responsible For Stop Codon Recognition Specificity

When one of three stop codons, UAA, UAG, or UGA, enters the ribosomal A site, translation termination occurs. Termination of translation in eukaryotes is mediated by two polypeptide chain release factors, eRF1 and eRF3, but the mechanism of polypeptide chain release factors recognizing stop codons remains unclear to date. In eukaryotes, eRF1 decodes three stop codons, wherease ciliates eRFl displays specificity of stop codon recognition, decoding one or two of three codons in ciliates cells.Many studies have shown that N domain of eRFl is responsible for the recognition of stop codons, where three highly conserved regions, GT motif, TASNIKS motif and YCF motif play important role in the process, however, the detail molecular mechanism of code determination by eRFl is still unclear. Then whether the specificity of stop codon recognition by ciliates eRF1 are regulated by three motifs or some key residues in this area, and the structural relationship among three motifs are valuable questions to be answered. This will administer to elucidating the molecular mechanism of eRF1 decoding stop codons.Firstly, we constructed a domain chimeric Tt/Sc eRFl that contained the eRF 1 N domain of Tetrahymena and the M, C domain of yeast eRF 1 of from Saccharomyces cerevisea. The characteristics and activity of chimeric eRF1 were analyzed by using the dual-luciferase read-through assay in eRF1 knockout yeast strain YDB447.We found that Tt/Sc eRF1 recognized three stop codons with specificity similar to that of eRF1 in Tetrahymena, displaying the higher readthrough levels at UAA(10.12%) and UAG(4.07%) than that at UGA(2.49%), significantly. To identify the key motif and amino acids residues in this domain, we constructed Tt/Sc eRF1 mutations by site-directed mutagenesis, changing the key amino acids of in the conserved motifs of Tetrahymena eRF1 N domain into the corresponding amino acids of S. cerevisea eRF1. Readthrough measurement revealed that readthrough level of mutations K57T/T59S/D63S in TASNIKS motif are increased 13.45%, 1.84%, respectively, at UGA, UAG, but reduced 3.48% at UAA, while readthrough level of mutations F125L/S128N in YCF motif at UGA was increased 3.04%, and reduced 1.89%,6.72% at UAG and UAA, respectively, suggesting changes of the amino acid in the conservative motif of Tetrahymena eRF1 had some impacts on the activity of chimeric eRF1.To analyse the role of conservative motifs and coordination among them in determination of eRF1 character, we constructed the intra-N-domain chimeras eRF1 using the method of bridging PCR to introduce conservative motifs and its adjacent amino acids of ciliate eRFl into corresponding position of Sc eRF1. The eRF1 from Giardia lamblia and Tetrahymena were used to construct the intra-N-domain chimeras eRF1, and resulted in the following mutants eRF1:G1(1-84aa)/Sc(76-149aa)-N, Sc(1-75aa)/G1(79-157aa)-N; Tt(1-77aa)/Sc(76-149aa)-N Sc(1-75aa)/Tt(78-151aa)-N; Sc(1-38aa)/Tt(41-77aa)/Sc(76-149aa)-N, Tt(1-40aa)/Sc(39-75aa)/Tt(78-151aa)-N, where the 1-40 aa contained GTx motif,41-77aa contained TASNIKS motif,78-151 aa contained Y-C-F motif. And thses intra-N-domains combined with M and C domain of yeast eRF1 were cloned into yeast expression vector pDB0948, meanwhile, some key amino acids residues were changed into that of yeast eRF1 in intra-N-domain chimeras eRF1. Readthrough assays and plasmid shuffle methods were used to analyse the function of these chimeras and mutant eRFl. The results indicated that, for chimeras of Glamblia and S. cerevisea eRFl, the readthrough level of Gl/Sc-N increase 12.80% and 0.81% at UGA and UAG respectively, whereas the readthrough level of Sc/Gl-N reduce 3.03%,2.92%,5.69% at UGA, UAG, UAA, respectively, indicating that the TASNIKS motif was related to the discrimination of three stop codons, and the Y-C-F motif inflenced the discrimination of second basic group. For chimeras of Tetrahymena, and S. cerevisea eRF1, the readthrough the efficiency of Tt/Sc-N eRF1 were very high at UGA, UAG and UAA (26.39%,8.02% and 10.75% respectively), whereas, the readthrough efficiency of Sc/Tt-N eRFl was relatively low,3.97%,2.09% and 3.20%, respectively, suggesting GTx and NIKS motif determine the recognition of UAG and UAA by eRF1.Altogether, we considered that motif GTx and NIKS were involved in the discrimination of the first U and second A in stop codons UAA and UAG, and Y-C-F motif determined the decoding of the second G in UGA. The results of this study provided basic data for elucidating the molecular mechanism of termination codon recognition by eRF1.