Mechanism Of Stop Codon Recognition Of Class I Polypeptide Release Factor

Termination of translation in eukaryotes is mediated by two interacting polypeptide chain release factors, eRF1and eRF3. While eRF1decodes the three stop codons, UAA,UAG and UGA, eRF3facilitates polypeptide chain release from the ribosome in a GTP-dependent manner. Eukaryotic translation termination typically requires the decoding of one of three termination codons UAA, UAG or UGA by polypeptide chain release factor eRF1. Stop codon reassignments have occurred very frequently in ciliates.Current models of stop codon decoding include linear models positing that stop codons physically bind to the TASNIKS motif in N-domain of eRF1, and ’cavity’ models proposing that the body of the N-domain of eRF1contains three cavities that can physically accommodate the first, second and third nt of the stop codons, respectively. Later, Kisselev and colleagues studied the effect of mutations in residues of N domain of eRF1that are conserved in standard code organisms but have diverged in many variant-code organisms (ciliates), leading the proposal of nonlinear model in which stop codon recognition is modulated by positive and negative determinants. More evidence for cavity model derived from evolutionary, mutagenesis and biochemical studies have been displayed recent years. More residues around the cavities involved in stop codon decoding and maintaining overall structural frame-work of motifs in N-domain were identified.Since previous researches revealed that N-terminal domain of eukaryote eRF1is responsible for stop-codon recognition, determinants should exist in the N domain of eRF1s that restrict their stop-codon specificity. Variant code species frequently alter this pattern of stop codon recognition, such as some ciliates species. Some class-specific residues in N domain of eRF1were identified and were considered to be responsible for the stop codon reassignment in these species.In the current study, the chimeric eRFls, which contains N domain of eRF1from Eo-eRF1b and eRF1(Bj-eRF1) fused with M and C domain of yeast eRFl, were designed to identify the key residues within the N domain of eRFl. We carry out the assays using the N domain of eRF1s from Euplotes octocarinatus (Eo-eRFlb) and Blepharisma japonicum(Bj-eRF1) as well as site-directed mutagenesis to identified the key amino acids residues and motifs involving in stop codon decoding.Then our experiments focus on viability assay of stop codon recogniton in Euplote eRFlb, Blepharisma eRF1and its mutations. To observe the function of chimeric eRF1proteins, plasmid shuffle procedure was carried out to assess whether these chimeric and mutant eRF1s could support the viability of yeast cells as the sole source of eRF1. The results displayed that Bj/Sc eRF1can complemented the sup45△mutation at30℃and22℃, while the cells grow sick in37℃, indicating the Bj-eRF1is temperature sensitive proteins in yeast cell. Most of yeast strains can grow with the support of these mutants Bj/Sc eRFls as the sole source of eRF1. In contrast, the Eo/Sc eRFlb were unable to support growth when present as the only source of eRF1in the cells in all temperature.Activity assay of stop codon recogniton in Euplote eRF1b, Blepharisma eRF1and its mutations is followed. We carry out the assays using the N domain of eRF1s from E. octocarinatus (Eo-eRF1b) and B. japonicwn (Bj-eRF1) as well as site-directed mutagenesis to identified the key amino acids residues and motifs involving in stop codon decoding by using dual-luciferase read-through assay system in eRF1knock-out yeast strains YDB447. Consistence with previous reports, three pockets in N domain of eRFls are involved in the decoding of three bases of stop codons, whereas allocation of the residues to each pocket are revalued according to our data. It is suggested that class-specific surface residues and hydrophobic residues function in different ways, the former directly interact with the base of stop codons, and the later might modulate the conformation of three pockets. The absolute conserved residues across all eRFls are responsible the relative orientation of three pockets corresponding to three bases of termination code.Summarizing the previous data and those obtained in this study, we have estimated that eRF1pocket3are consisted of residues G31, T32,V66, N67, S70, and V71is proximal to the third base of the stop codon. The changes of these residues remarkably affect the stop codons discrimination of eRF1, suggesting two or three residues of this peptide may be involved in discrimination of A or G of stop codon. Pocket2made up of residues L126, C127, D128and H132with floor residue V110, and is associated with the discrimination of second base of stop codon by D128or H132, respectively. The base A-N6in the second position of stop codon is decoded by D128, and the decoding process is modulated by C127. The base G in second position is decoded by H132. and the this process is modulated by L126. Pocket1is a deep pit lined with hydrophobic residues (L37,139, V48, and L82), and bounded by residues R47, V48, M51of alpha-helix2and S123, L124, Y125of the beta4-strand. S123is considered to interact with U by potential hydrogen bonding between hydroxyl group of S123and U-O2. Qur quantitative analysis of collection of point mutants in variant eRF1s from ciliates and yeast have not conclusively revealed the certain correlation between evolutionary conservation of class specific residues in eRFls and termination-related functional specificity of eRF1, and are limited for elaborating the detailed mechanism of stop codon reassignment in ciliates.The explanation of the eRF1specifically bind only the three codons, UAA, UAG, and UGA, and exclude the UGG tryptophan sense codon was proposed by Betram et al.. Here, we propose that UGG theoretically can be recognized by eRF1, however, the tryptophan tRNA with anticodon CCA in cells can preferentially recognize UGG codon, leading to no opportunity for eRF1to meet with UGG. Like the suppressor tRNA exist in the ciliates cell, the corresponding stop codon (e.g. UGA in Euplotes) can not be decoded by eRF1. Mechanism of stop codon recognition of class I polypeptide release factor or termination codon reassignment are problems that have not yet been fully clarified, and should be further in-depth discussed.