Sequence And Functional Analysis Of Aminoacyl-tRNA Synthetase Of Euplotes Octocarinatus

Aminoacyl-tRNA synthetases?aaRSs?ligate specific amino acids and the corresponding tRNAs,they translate the nucleic acid language into the amino acid language and thereby known as the“second genetic code”.Normally,for each of the 20 standard amino acids cells are expected to express at least one aaRS.Based on the conserved architecture of the catalytic core domains,aaRS families are divided into two structurally distinct classes?class I and II?.All known aaRSs are multidomain proteins with complex modular architectures.AaRSs also perform several non-canonical functions in transcription,translation,apoptosis,angiogenesis and inflammation by different mechanisms.Euplotes octocarinatus is a classical single-cell protozoa with high evolutionary status.Euplotes exhibits nuclear dimorphism:a germline micronucleus?MIC?and a somatic macronucleus?MAC?,and its termination codon UGA,as sense codon,encodes cysteine or selenocysteine.In addition,a previous study showed that approximately 11%of the genes in E.octocarinatus employ+1 PRF in the process of protein translation.However,its molecular mechanism is still unclear.Considering that aaRSs can provide the essential substrates for protein synthesis and maintain or control translation fidelity.Therefore,this study firstly performed a comprehensive analysis and identification on type,number,structure and origin of aminoacyl-tRNA synthetase genes from Euplotes octocarinatus?EoaaRS?using the bioinformatics methods.Furthermore,in order to explore the influence of tRNA^Lys-1ys-1 with expanded anticodon loop on the typical+1 PRF with the frameshift motif“AAA-UAR”,EoLysRS-1 was purified by affinity chromatography,the tRNA^Lysys was prepared by in vitro transcription and the firefly luciferase frameshift reporter plasmid pFluc-FS was constructed.Then we investigated the molecular mechanism of+1 PRF using TNT^?Quick Coupled Transcription/Translation systems.1.Identification and analysis of the EoaaRS genes.The amino acid sequences of HsaaRSs were used as the reference sequences and a series of BLAST searches were performed.Finally,a total of 45 EoaaRS genes encodeing 20 different kinds of EoaaRS were identified.Most EoaaRSs were encoded by multiple genes except EoGlnRS and EoAlaRS.According to the subcellular localization analysis,only eight EoaaRSs corresponding to six kinds of EoaaRS had mitochondrial targeting peptides.In addition,there are several EoaaRSs utilizing+1/+2 PRF to produce complete protein products.Domain analysis demonstrated that some EoaaRSs contained several extra domains,which implied that they may perform new functions beyond aminoacylation.Evolutionary analysis revealed that both cytoplasmic EoLysRS-1 and mitochondrial EoLysRS-2 originated from bacteria,while two cytoplasmic EoGlyRSs originated from archaea,which indicating their evolutionary complexity.2.Cloning and sequence analysis of the EoLysRS genes.The EoLysRS-1 and EoLysRS-2 were obtained by PCR,and their open reading frames?ORF?were 1800 bp and 1533 bp,respectively.Moreover,EoLysRS-2 had three introns of GT-AG type and was identified as+2 PRF candidate genes with slippery sequence“AUA-UAG”.Compared to EcLysRS,the N-terminus of EoLysRS-1 has an extended peptide segment rich in lysine and arginine.It has been reported that the unique N-terminal extension domain in eukaryotic organisms can increase the aminoacylation efficiency of LysRS.3.Expression,purification and thermal stability of EoLysRS-1.To expression the EoLysRS-1 in Escherichia coli,three TGA codons were mutated to TGT codon which encoding cysteine in E.coli.The recombinant plasmid pET-28a-LysRS-1 was constructed and induced to express in the E.coli Transetta?DE3?expression strain.The recombinant protein EoLysRS-1was purified by Ni²⁺-NTA affinity chromatography and confirmed by Western blotting.We calculated dissolution temperature?T_m?of EoLysRS-1was 65?by circular dichroism.4.Construction of the in vitro translation examination system for+1programmed ribosomal frameshifting of E.octocarinatus.The firefly luciferase frameshift reporter plasmid pFluc-FS containing the T7 promoter was constructed.The activity of the luciferase was detected in a rabbit reticulocyte in vitro coupled transcription/translation system with the EoLysRS-1 and tRNA^Lysys obtained by in vitro transcription.The results showed that either the normal tRNA^Lys-2ys-2 or the tRNA^Lys-1ys-1 with an expanded anticodon loop had no significant effect on luciferase activity.In summary,45 EoaaRS nanochromosomes encoding 20 different kinds of EoaaRS were identified by bioinformatics.There were two LysRS genes in E.octocarinatus.Unlike the mitochondrial EoLysRS-2,the cytoplasmic EoLysRS-1 has a eukaryotic characteristic N-terminal extension.The fusion protein EoLysRS-1 was purified by Ni²⁺-NTA affinity chromatography and the T_m value of which was 65?.The firefly luciferase frameshift reporter plasmid pFluc-FS was constructed.In in vitro transcription/translation coupled rabbit reticulocyte lysates,the effect of tRNA^Lys-1ys-1 with expanded anticodon loop on the+1 PRF was investigated using the firefly luciferase frameshift reporter plasmid pFluc-FS.These findings provide experimental data for further understanding of the function of EoaaRSs and for exploring the molecular mechanism of the programmed ribosome frameshift.