Structural And Functional Analysis Of N And C Domain Of The Class â…  Polypeptide Release Factors In Euplotes Octocarinatus

The process of protein synthesis is terminated by one of the three stop codons, UAA, UAG, or UGA, which are recognized by proteins called class I peptide release factors. In eukaryotes, a single release factor, eRF1, recognizes all the three stop codons. Upon stop codons recognition, eRF1s catalyze hydrolysis of the ester bond that links the nascent polypeptide chain to P-site tRNA, and thereby induce peptide release. Subsequently, eRF3s remove the eRF1from the ribosome.Most eukaryotes possess a single Class I peptide release factor eRF1. eRF1proteins from eukaryotic species share significant homology at the primary amino acid sequence level. The human eRFl structure, in a crystal structure and in solution, consists of three domains (N, M and C domains). N domain binds the stop codon directly in the ribosomal A-site. M domain interacts with the peptidyl transferase center leading to the hydrolysis of the last peptidyl-tRNA bond through the GGQ motif. C domain is the binding site upon eRF3. Both release factors eRF1and eRF3are key components of the termination process. eRF3may act as a proofreading factor by coupling stop codon recognition by eRFl to efficient polypeptide chain release.Some organisms which deviate from the standard genetic code are called variant code organisms. For example, the ciliate species retain UAA and UAG as stop codons, but reassign UGA to sence codons. In Euplotes octocarinatus, UGA was reassigned as cysteine, while UGA was reassigned as tryptophan in Blepharisma japonicum. eRF1s from variant code organisms retain substantial overall amino acid sequence homology with eRF1s from standard code organisms, particular variations may account for the functional differences in these proteins. The highly conserved amino acid motifs in standard code organisms, which are more degenerate in variant code organisms, may represent key residues that mediate recognition to stop codons. Different from most eukaryotes, Euplotes octocarinatus possesses two Class I peptide release factors, Eo-eRF1a and Eo-eRF1b. The E. octocarinatus QRF1b gene contains three UGA stop codons recoding cysteine. UGAs were mutated as sense codon for expressing in vitro. In this study, the N and C domain of Eo-eRF1a, Eo-eRF1b and Bj-eRF1were cloned, expressed, purified and identified. It was previously shown that a short portion of the C-terminal domain of eRF3(Eo-eRF3Cm6) was sufficient for Eo-eRFla binding in E. octocarinatus. In this research, ciliate eRF1and its C domains were proved binding with Eo-eRF3Cm6by in vitro pull down assay.Most proteins possess chromophoric groups. The changes of protein endogenous fluorescence spectrometry could reflect the conformation change of these proteins. In this research, each N or C domain of ciliate eRF1(Eo-eRF1a, Eo-eRF1b and Bj-eRF1) possesses a highly conserved tryptophan. The results from fluorescence spectrometry showed that the highly conserved tryptophan side-chain (W-11) in eRF1N domain located in hydrophilic environment, while the highly conserved tryptophan side-chains in eRF1C domain (W-383in Eo-eRF1a C, W-373in Eo-eRF1b C and W-373in Bj-eRF1C) located in more hydrophobic environment. In comparing the relative fluorescence profiles of eRF1s and that of binding with Eo-eRF3Cm6, the spectral peak maximum was blue shifted from340nm to330nm. This result suggested that the interaction of the two proteins changed the conformation of eRF1s. The C-terminal domain of eRF1is known to interact with the C domain of eRF3. But, comparing the relative fluorescence profiles of Eo-eRF1bC and the complex Eo-eRF1bC/Eo-eRF3Cm6, the spectral peak maximum has no visible change. The data of fluorescence quenching show that the quenching constant and fraction decreased. These results show the fluorophores in eRF1s turned more hydrophobic, and the conformation changing were driven by this binding. These results suggested that the eRF1N domain interacts with its C domain within intact protein.In order to reveal the binging sites on eRF1-eRF3interaction, the N and C domain of Eo-eRF1a/b and Bj-eRF1were analyzed by computational simulation basing on homolog modeling. And the possible binding sites were tested in exprements subsequently.Analysis by computational simulation demonstrated that several peptides involved in Eo-eRF1/EoeRF3interaction directly. Among these peptides, the two motifs GVEDT and GFGG were highly conserved.1290and D293(V294å'ŒD297in Bj-eRF1) of Eo-eRF1were also highly conserved. Site-directed mutagenesis was introduced, then mutants biding with Eo-eRF3Cm6were tested by pulldown and Western blotting. The results were used to assess the contribution of mutants to eRF1-eRF3interaction. The major interaction means were also dissculsion. In Bj-eRF1, V294, corresponding to Ile (1290in Eo-eRF1a/b) in other species, was reversed by site-directed mutagenesis. When binding with Eo-eRF3Cm6, the mutant Bj-eRF1C V294I displayed a similar ability to the wild Bj-eRF1C When this site(V294in Bj-eRF1,1290in Eo-eRF1a/b) mutanted to Ala, Eo-eRF1a/b C1290A and Bj-eRF1C V294A exhibit a lower binding capacity with Eo-eRF3Cm6. So this site is a key site in eRF1-eRF3interaction. D293, another highly conserved site on Eo-eRF1C (D297in Bj-eRF1), was conformed playing an important role in eRF1-eRF3interaction. Compared to the wild Eo-eRF1C, the two mutations D293N and D293V of Eo-eRF1aC exhibit a similar binding capacity, while D293N and D293V of Eo-eRF1bC exhibit the decreased level in binding with Eo-eRF3Cm6. Eo-eRF1a and Eo-eRF1b may select different manners to interact with Eo-eRF3. These studies contribute to the better understanding the mode of eRF1-eRF3interaction. In our research, the results from computational simulation suggested that the nonbonded interaction is predominant interaction. But, in Eo-eRF1aC complex, the electrostatic interaction is greater than that of Eo-eRF1bC and Bj-eRF1C complex. From the above, we can draw a conclusion that Eo-eRF1aC, Eo-eRF1bC, and Bj-eRF1C can interact with Eo-eRF3Cm6. D293in Eo-eRF1bC and D297in Bj-eRF1C domain were conformed playing an important role in eRF1-eRF3 interaction. When binding with EoeRF3Cm6, Eo-eRF1bC shares similar characteristics with Bj-eRF1C. But the Eo-eRF1aC and Eo-eRF1bC interact with Eo-eRF3Cm6with different manner.The structures of the mutations of eRF1N domain have been investigated by modeling, molecular dynamics simulations and conformational analysis. Although the electrostatic energy and hydrogen bonds changed distinctly at mutant sites, these changes can’t connect with reversion of eRF1function. A flexible loop (95-103) was noticed for its special spatial situation, which is centered between TASNIKS motif and C-domain of eRF1. This loop was deduced to involve in stop codons recognition.