guanosine-triphosphate and sordarin

The Intergenic Region Internal Ribosome Entry Sites (IGR IRESs) of Discistroviridae promote protein synthesis without initiation factors, with IRES translocation by elongation factor 2 (eEF2) being the first factor-catalysed reaction. Here, we developed a system that allows for the observation of intersubunit conformation of eukaryotic ribosomes at the single-molecule level by labeling rRNA. We used it to follow translation initiation and subsequent translocation of the cricket paralysis virus IRES (CrPV IRES). We observed that pre-translocation 80S-IRES ribosomes spontaneously exchanged between non-rotated and semi-rotated conformations, but predominantly occupied a semi-rotated conformation. In the presence of eEF2, ribosomes underwent forward and reverse translocation. Both reactions were eEF2 concentration dependent, indicating that eEF2 promoted both forward and reverse translocation. The antifungal, sordarin, stabilizes eEF2 on the ribosome after GTP hydrolysis in an extended conformation. 80S-CrPV IRES-eEF2-sordarin complexes underwent multiple rounds of forward and reverse translocations per eEF2 binding event. In the presence of sordarin, neither GTP hydrolysis nor a phosphate release were required for IRES translocation. Together, these results suggest that in the presence of sordarin, eEF2 promotes the mid and late stages of CrPV IRES translocation by unlocking ribosomal movements, with mid and late stages of translocation being thermally driven.

Topics: Guanosine Triphosphate; Internal Ribosome Entry Sites; Peptide Elongation Factor 2; Protein Biosynthesis; RNA, Viral

Sordarin derivatives are selective inhibitors of fungal protein synthesis, which specifically impair elongation factor 2 (EF-2) function. We have studied the effect of sordarin on the ribosome-dependent GTPase activity of EF-2 from Candida albicans in the absence of any other component of the translation system. The effect of sordarin turned out to be dependent both on the ratio of ribosomes to EF-2 and on the nature of the ribosomes. When the amount of EF-2 exceeded that of ribosomes sordarin inhibited the GTPase activity following an inverted bell-shaped dose-response curve, whereas when EF-2 and ribosomes were in equimolar concentrations sordarin yielded a typical sigmoidal dose-dependent inhibition. However, when ricin-treated ribosomes were used, sordarin stimulated the hydrolysis of GTP. These results were compared with those obtained with fusidic acid, showing that both drugs act in a different manner. All these data are consistent with sordarin blocking the elongation cycle at the initial steps of translocation, prior to GTP hydrolysis. In agreement with this conclusion, sordarin prevented the formation of peptidyl-[(3)H]puromycin on polysomes from Candida albicans.

Topics: Antifungal Agents; Candida albicans; Dose-Response Relationship, Drug; Fusidic Acid; GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Triphosphate; Hydrolysis; Indenes; Models, Biological; Peptide Chain Elongation, Translational; Peptide Elongation Factor 2; Peptide Elongation Factors; Protein Synthesis Inhibitors; Puromycin; Ribosomes; Ricin