The Influence Of Polypeptide Chain Release Factor ERF1 On Programmed Translational Frameshifting

Programmed translational frameshifting is a phenomenon whereby the ribosome is guided toward a triplet nucleotide that is either shifted one nucleotide position upstream (+1 frameshift) or one nucleotide position downstream (-1 frameshift). Programmed translational frameshifting has been identified in a broad range of organism genes. According to statistics, among all of the organisms the Euplotes genes harbor a highest frequent frameshifting except virus. A recent analysis of gene sequences available in GenBank from ciliates in the genus Euplotes indicates that >5% required one or more+1 translational frameshifts to produce their predicted protein products. Several factors can influent the effect of frameshift, including low concentration of release factors, SD-like sequences, the secondary structures and so on. Human peptide chain release factor (eRF1) affects-1 programmed translational frameshifting of the HIV-1 virus. Morever, eRF1 also has a significant impact on the+1 programmed frameshifting that occurs at a high frequency in euplotes.To further investigate the key sequence of eRF1 which affected the programmed frameshifting and regulation mechanism, a dual luciferase report system in yeast has been established and hybrid peptide chain release factor Eo/Sc eRF1 was constructed with the N domain of Euplotes octocarinatus eRF1b and MC domains of Saccharomyces cerevisiae eRF1, then frameshift efficiency was measured in the presence of various mutations of Eo-eRF1b N domain.1. Differents of shifty stop frameshift sequences were inserted into a dual luciferase reporter vector. Meanwhile, plasmid shuffle procedure was used to screen strains YDB447/pDB967. The efficiency of programmed frameshifting on shifty stop frameshift sites was measured in the presence of variant forms of eRF1. As a result, the dual luciferase report system for studying programmed translational frameshifting in yeast has been established successfully.2. To investigate the connection between stop codon reassignment and frequently use of shifty stop frameshifting, hybrid peptide chain release factor Eo/Sc eRF1 was constructed with the N domain of Euplotes octocarinatus eRF1b and MC domains of Saccharomyces cerevisiae eRF1, then frameshift efficiency was measured in the presence of various mutations of Eo-eRF1b N domain. Compared with the Eo/Sc eRFl, UAA/UAG frameshift efficiency significant increased in the presence of Eo/Sc eRF1b G31S/T32R, it showed that the conserved GT motif in eRF1 involved in the regulation of programmed translational frameshifting. I126L in Eo/Sc eRF1b mutants decreased frameshifting at UAA/UAG. Compared with YLCDNKF of yeast, unique YICDNKF enhanced the frameshift efficiency at AAA-UAR-V by reduced the recognition of UAA/UAG in euplotes.3. The effectiveness of the dual luciferase report system was affected by inserted frameshift gene into dual luciferase reporter vector. Frameshift gene Ndr2 from Euplotes octocarinatus were inserted into a dual luciferase reporter vector, and the efficiency of programmed+1 frameshifting on Ndr2 was measured in the presence of variant forms of eRF1. Eo/Sc eRF1 increased the frameshifting at Ndr2 effectively.Therefore, unique YCF motif may be one of the reasons that caused the high programmed translational frameshifting frequency in euplotes. These results provide experimental data for further studies of the molecular mechanisms of programmed translational frameshifting in ciliates.