Analysis On The Specificity Of Stop Codon Recognition Of Ciliates ERF1

In eukaryotes a single class-1 translation termination factor eRFl decodes three stop codons:UAA,UAG and UGA. Variant codon species, such as protozoan ciliates, frequently exhibit altered stop codon recognition. Earlier studies have identified that some class-specific residues in the eRF1 N-terminal domain play a critical role in decoding each stop codon. And substitutions of these amino acids may affect on the function of eRFl decoding stop codon, and generation of the specific function of eRF1 can cause stop codon reassignment in these organism. Recent studies demonstrated that the highly conserved "TASNIKS" and "YxCxxF" motifs in N terminal domain of eRF1 play important roles in stop codon recognition for eRFl, but these conclusion is challenged by recent studies. The effect of these conserved motifs on the specificity of eRFl toward stop codon are variant depending on different assay system and eRFl gene from different ciliates. In addition, post-translational modifications of eRF1 affect the function of eRFl, and whether post-translational modification affect on the specificity of stop codon recognition of ciliates eRFl are valuable projects. The aim of this research is to analyze the function of motifs of eRFl in stop codon recognition process by using the constructions consisting of intra-N-domain chimeras harboring motifs from cialiates eRFl and S. cerevisiae eRF1; the second aim of our experiment is to analyze the effect of phosphorylation modification of eRFl on specifity of stop codon recognition of eRFl by introducing or deleting the phosphorylated kinase recognition sites of ciliates eRF1s.Firstly, we constructed the domain chimeric Bj/Sc eRFl and Eo/Sc eRFl, in which N-terminal domain of Blepharisma japonicum or Euplotes octocarinatus eRF1 fused to M and C domains of S. cerevisiae eRF1. The intra-N-domain chimeras eRFl as following were contructed by bridging PCR, Bj(1-76aa)/Sc(76-149aa)-N, Sc(1-75aa)/Bj(77-152aa)-N; Eo(1-74aa)/Sc(76-149aa)-N,Sc(1-76aa)/Eo(75-152aa)-N; Sc(1-38aa)/Eo(38-74aa)/Sc(76-149aa)-N,Eo(1-37aa)/Sc(39-76aa)/Eo(75-152aa)-N, where the fragment 1-38aa harbors "GTx" motif, the fragment 38-74aa harbors "TASNIKS" motif, and the fragment 75-152aa harbors "YxCxxF" motif. The functions of intra-N-domain chimeras eRFl were measured by using dual luciferase readthrough report assays and plasmid shuffle procedure in yeast cells. The results suggested that the "GTx" and "TASNIKS" motif of B. japonicum eRFl are involved in decoding three stop codon, and "YxCxxF" motif are important for decoding the second base G of UGA. However, Intra-N-domain chimeras with E. octocarinatus eRFl motifs have little effect on the stop codon specificity. The further study revealed that "TASNIKS" motif might be determinant of UAA and UAG specificity of eRF1, and "YxCxxF" motif did not contribute to the stop codon specificity of eRF1. In conclusion, the conformation of the motif and the amino acid position determine stop codon recognition specificity of eRFl, and to some extent support the "cavity" model, a hypothesis of eRFl decoding stop codons.Secondly, post-translational modification of proteins has a great influence on interaction between macromolecules in cells. Some amino acids, Ser and Thr in the context of sequences, YGKSSNIKS, SALTSTKER, CEKKYTIDF, and NTTLYICDN, in the Bj eRF1 N-terminal domain are predicted to be phosphorylated by kinase, which are possible class-specific residues responsible for codon discrimination. Whether the modifications of eRF1 affect on the stop codon discrimination. The recognition sites of protein kinases were changed by mutagenesis to investigate the effects on stop codon discrimination by phosphorylation. Our findings indicated that some amino acids substituted of eRFl significantly affect on stop codon selectivity.Finally, our research reveal that post-translational modifications of proteins also occurs in prokaryotes, at least phosphorylarion modification. We expressed the C-terminal domain of B. japonicum eRFl in Escherich coli, and the purified protein were treated with phosphotase. Analysis by using native polyacrylamide gel electrophoresis and the western blotting indicated that the C-terminal domain of B. japonicum eRFl were phophorylated suggesting there existed at least one or more phosphorylation sites in C terminal domain of B. japonicum eRF1.