| Research Abstract |
Translation termination in eukaryotes is mediated by two eukaryotic release factors, eRF1 and eRF3. eRF1 recognizes all three stop codons and induces polypeptide release, while eRF3 binds to eRF1 and participates in translation termination though the regulatory role of eRF3 is still unknown. Importantly, eRF3 interacts with various proteins of distinct biological functions. Here, we investigated the effect of these binding factors on functionality and stability of eRF3 using a temperature-sensitive mutant eRF3ts, which is susceptible to factor binding to change the growth phenotype or cellular protein level. Of factors tested, Itt1 over-expression and Sla1 knockout severely impaired viability of eRF3ts cell and its protein abundance in permissive and semipermissive conditions. Sla1 over-expression reversed the phenotype. It is reported that Itt1 and Sla1 bind to the N-terminal extension domain (NED) of eRF3, unlike the other no-effect factors that bind to the C-terminal domain (CTD). A
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lthough NED itself is dispensable, NED-less eRF3ts altered in the stability and functionality. Moreover, Itt1-induced eRF3ts lethality was significantly restored by pep4, prb1 and prc1 knockouts that are defective in vacuolar proteolysis. These findings suggest that NED functions to switch the functional mode of eRF3 depending on the nature of binding factors.
Wyosine and its derivatives, such as wybutosine, found in eukaryotic and archaeal tRNAs, are tricyclic hypermodified nucleosides. In eukaryotes, wybutosine exists exclusively in position 37, 3'-adjacent to the anticodon, of tRNAPhe, where it ensures correct translation by stabilizing the codon-anticodon base pairing during the ribosomal decoding process. Recent studies revealed that the wyosine biosynthetic pathway consists of multistep enzymatic reactions starting from a guanosine residue. Among these steps, TYW1 catalyzes the second step to form the tricyclic ring structure, by cyclizing N1-methylguanosine. In this study, we solved the crystal structure of TYW1 from Methanocaldococcus jannaschii at 2.4 A resolution. TYW1 assumes an incomplete TIM barrel with(/)6 topology, which closely resembles the reported structures of radical SAM enzymes. Hence, TYW1 was considered to catalyze the cyclization reaction by utilizing the radical intermediate. Comparison with other radical SAM enzymes allowed us to build a model structure complexed with S-adenosylmethionine and two [4Fe-4S] clusters. Mutational analyses in yeast supported the validity of this complex model structure, which provides a structural insight into the radical reaction involving two [4Fe-4S] clusters to create a complex tricyclic base. Less
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