Functional Characterization Of Polypeptide Release Factor 1b From The Euplote Octocarinatus

In the peptide chain elongation process, when the stop codon enters the ribosome A site, there is no corresponding amino acid-tRNA binds to the peptide chain. However, release factor (RF) can identify and bind to stop codon, then hydrolysis the ester bond. Two interacting polypeptide release factors (RFs) are required to complete protein biosynthesis. One is codon-specific classâ… release factor, which is responsible for stop codon recognition and promotes hydrolysis of the ester bond linking polypeptide chain with the peptidyl site tRNA; the other is classâ…¡release factor, which acts as a GTPase. The energy released by hydrolysising GTP promotes the release of newly synthesis peptide from ribosome.Euplotes Octocarinatus, one of simplest unicellular eukaryotes, has a special position in evolution. Different from other eukaryotes, two genes encoding classâ… release factor are cloned from its macronucleus, named eRF1a and eRF1b, respectivily. American scientists have done a great many of experiment on eRF1a. In order to deeply understand the mechanism of termination of protein biosynthesis, analysis the possible relationship between these two RF1s, and investigate the function of eRF1b, we used eRF1b of E.octocarinatus as centers to carry out experiments in this study.In the first place, the total mRNA was extracted from Euplotes and then reversed them to cDNA. The relative expression level of eRF1a and eRF1b on mRNA level were compared by Real-time PCR, and the result showed that the expression level of eRF1b was 16-fold higher than that of eRF1aSince the interaction of two release factors is precondition to ensure rapid and effective release of newly synthesis protein, we used yeast-two hybrid assay to detect the interaction between eRF1b and eRF3. Because there are three stop codons decoding cysteine in Euplotes eRF1 gene, mutagnesis of the UGA to UGC have been conducted in order to ensure this gene could normally expresses cysteine in E.coil and yeast. The results showed that the interaction indeed existed between eRFlb and eRF3. Combined with first part of the experiment, we suggested that eRF1b might play more important role than eRF1a. On the other hand, yeast two hybrid method also confirmed the interaction between two types of release factors from Giardia lamblia. It provided more data to elucidate the relationship between evolution of release factors and biological evolution.To further detect whether the function and functional sites are similar between eRF1a and eRF1b in Euplote cell, co-localization analysis have been performed by using the macronuclear artificial chromosome harboring the gene of green fluorescence gene (EoMAC_G) and red fluorescence gene (EoMAC_R). Firstly, we cloned eRF1a and eRF1b into the EoMAC_G and EoMAC_R, respectively. Then we co-transformed these two recombinant plasmids into Euplotes cell and observed the distribution of two release factors in Euplotes cell by using fluorescent microscope and confocal microscope. The results indicated that both eRF1s mainly distributed on the inner side of the macronucleus. This is consistent with the distribution of the endoplasmic reticulum that connects to the nuclear membrane to generate a field for protein biosynthesis. The shared localization indicated that they probably have the same function in cells, i.e., protein biosynthesis.Based on the above experiments, we utilized yeast strain knockout sup45 and dual luciferase readthrough assay to detect the recognition ability of eRFlb. Firstly, we subcloned the eRF1b from Euplotes into the plasmid harboring gene of M and C domain from sup45 to yield the hybrid plasmid expression Eob/Sc eRFlb. Secondly, this hybrid plasmid was transformed into yeast strain YDB447 containing plasmid pUKC802 carried wild type sup45, and then scribed the yeast onto 5-fluoroorotic acid (5-FOA) plate. The results demonstrated that the hybrid protein Eob/Sc eRF1 could not support the viability of transformants as the sole source of eRF1 in yeast cells. This phenomenon indicated that eRFlb cannot recognize all three stop codons. In order to detect which stop codon the N domain of eRFlb can recognize, then we transformed this plasmid into 12 different yeast strains. The traits of these 12 yeast strains are that they contain dual-luciferase reporter genes, inserted by three different stop codons and relevant negative controls which contain sense codons. The activities of these two difference dual luciferase before and after stop codons indicated the recognition activities of class I release factor. The results suggested that eRF1b only recognized UAA and UAG as stop codons, just like eRF1 a. In order to investigate the important role of key amino acids in class I release factor N-domain during the process of stop codon recognition, through comparing the amino acids by bioinformatics method, we found that "GT" was a highly conserved amino acids motif in all organisms. Based on size and electric property of these two amino acids, we did three different site-directed mutations. The results showed that, as one of three highly conserved motifs in N domain, "GT" had key role in recognition ability. After mutations, the percent of readthough decreased sharply, these data proved that the "Cavity Model" might be the best hypothesis to explain the mechanism of termination of protein biosynthesis. Further bioinformatics comparison indicated that several special sites "V59, A70,I126" specifically emerged in organisms recognizing UAA and UAG as stop codons only. Thus, these three sites may also have crucial roles. Single and combined mutations proved they are also important in recognition.