title: Identification of amino acids responsible for stop codon recognition for polypeptide chain release factor.
abstract: One factor involved in eukaryotic translation termination is class 1 release factor in eukaryotes (eRF1), which functions to decode stop codons. Variant code species, such as ciliates, frequently exhibit altered stop codon recognition. Studies revealed that some class-specific residues in the eRF1 N-terminal domain are responsible for stop codon reassignment in ciliates. Here, we investigated the effects on stop codon recognition of chimeric eRF1s containing the N-terminal domain of Euplotes octocarinatus and Blepharisma japonicum eRF1 fused to Saccharomyces cerevisiae M and C domains using dual luciferase read-through assays. Mutation of class-specific residues in different eRF1 classes was also studied to identify key residues and motifs involved in stop codon decoding. As expected, our results demonstrate that 3 pockets within the eRF1 N-terminal domain were involved in decoding stop codon nucleotides. However, allocation of residues to each pocket was revalued. Our data suggest that hydrophobic and class-specific surface residues participate in different functions: modulation of pocket conformation and interaction with stop codon nucleotides, respectively. Residues conserved across all eRF1s determine the relative orientation of the 3 pockets according to stop codon nucleotides. However, quantitative analysis of variant ciliate and yeast eRF1 point mutants did not reveal any correlation between evolutionary conservation of class-specific residues and termination-related functional specificity and was limited in elucidating a detailed mechanism for ciliate stop codon reassignment. Thus, based on isolation of suppressor tRNAs from Euplotes and Tetrahymena, we propose that stop codon reassignment in ciliates may be controlled by cooperation between eRF1 and suppressor tRNAs.
authors.affiliation: a Key Laboratory of Chemical Biology and Molecular Engineering, Ministry of Education, China; and Institute of Biotechnology, Shanxi University, Taiyuan 030006, China.
authors.forename: Lijun
authors.initials: L
authors.lastname: Xu
authors.forename: Yanrong
authors.initials: Y
authors.lastname: Hao
authors.forename: Cui
authors.initials: C
authors.lastname: Li
authors.forename: Quan
authors.initials: Q
authors.lastname: Shen
authors.forename: Baofeng
authors.initials: B
authors.lastname: Chai
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Aihua
authors.initials: A
authors.lastname: Liang
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D018388:Codon, Terminator; D006801:Humans; D016296:Mutagenesis; D010454:Peptide Termination Factors / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D018388:Codon, Terminator; D010454:Peptide Termination Factors