真核释放因子eRF1,由ETF1基因编码,识别终止密码子并在翻译终止期间诱导肽释放。由于可变剪接,ETF1产生几种不同的转录本,可以形成两种eRF1同种型。同种型1编码经过充分研究的规范eRF1,同种型2比同种型1短33个氨基酸残基,完全未研究。使用重组的哺乳动物体外翻译系统,我们表明,人类eRF1的同工型2也参与翻译。我们表明eRF1iso2可以与核糖体亚基和终止前复合物相互作用。然而,其密码子识别和肽释放活性下降。此外,eRF1同工型2对UGA表现出单能性。我们发现eRF1同工型2与eRF3a相互作用,但刺激其GTP酶活性明显差于主要同工型eRF1。此外,我们在无细胞翻译系统中研究了eRF1亚型2对终止密码子连读和翻译的影响。我们观察到eRF1同种型2抑制了uORF的终止密码子连读并降低了长编码序列的翻译效率。基于这些数据,我们假设人eRF1亚型2可以参与翻译终止的调节。此外,我们的数据支持先前陈述的假设,即GTS环对于eRF1对所有终止密码子的多能性很重要.而N结构域eRF1的螺旋α1被认为参与了eRF3在GTP水解后发生的核糖体A位点的eRF1的构象重排,这确保了肽基tRNA在核糖体P位点的水解。
Eukaryotic release factor eRF1, encoded by the ETF1 gene, recognizes stop codons and induces peptide release during translation termination. ETF1 produces several different transcripts as a result of alternative splicing, from which two eRF1 isoforms can be formed. Isoform 1 codes well-studied canonical eRF1, and isoform 2 is 33 amino acid residues shorter than isoform 1 and completely unstudied. Using a reconstituted mammalian in vitro translation system, we showed that the isoform 2 of human eRF1 is also involved in translation. We showed that eRF1iso2 can interact with the ribosomal subunits and pre-termination complex. However, its codon recognition and peptide release activities have decreased. Additionally, eRF1 isoform 2 exhibits unipotency to UGA. We found that eRF1 isoform 2 interacts with eRF3a but stimulated its GTPase activity significantly worse than the main isoform eRF1. Additionally, we studied the eRF1 isoform 2 effect on stop codon readthrough and translation in a cell-free translation system. We observed that eRF1 isoform 2 suppressed stop codon readthrough of the uORFs and decreased the efficiency of translation of long coding sequences. Based on these data, we assumed that human eRF1 isoform 2 can be involved in the regulation of translation termination. Moreover, our data support previously stated hypotheses that the GTS loop is important for the multipotency of eRF1 to all stop codons. Whereas helix α1 of the N-domain eRF1 is proposed to be involved in conformational rearrangements of eRF1 in the A-site of the ribosome that occur after GTP hydrolysis by eRF3, which ensure hydrolysis of peptidyl-tRNA at the P site of the ribosome.