Studies of network of factors in translation

真核生物的翻译终止是由两个真核释放因子eRF1和eRF3介导的。ERF1识别所有三个终止密码子，并诱导多肽释放，而eRF3与eRF1结合并参与翻译终止，但eRF3的调节作用尚不清楚。重要的是，eRF3与多种具有不同生物学功能的蛋白质相互作用。在这里，我们使用一个温度敏感的突变体eRF3ts来研究这些结合因子对eRF3功能和稳定性的影响，eRF3ts对因子结合敏感，可以改变生长表型或细胞蛋白水平。在检测的因素中，Itt1过表达和Sla1基因敲除严重损害了eRF3ts细胞的活力及其在允许和半允许条件下的蛋白丰度。Sla1的过表达逆转了细胞的表型。据报道，Itt1和Sla1与eRF3的N-末端延伸域(NED)结合，不像其他与C-末端结构域(CTD)结合的无影响因子。A…尽管NED本身是可有可无的，但无NED的eRF3在稳定性和功能上发生了变化。此外，空泡蛋白降解缺陷的Pep4、prb1和Prc1基因敲除显著恢复了Itt1诱导的eRF3ts致死性。这些发现表明，NED的功能是根据结合因子的性质来切换eRF3的功能模式。真核和古生物tRNA中发现的Wyosine及其衍生物，如Wybuosine，是三环超修饰核苷。在真核生物中，Wybuosine只存在于tRNAPhe反密码子附近的37，3‘-位，在核糖体解码过程中，它通过稳定密码子-反密码子碱基对来确保正确的翻译。最近的研究表明，Wyosine的生物合成途径由从鸟苷残基开始的多步酶反应组成。在这些步骤中，TYW1通过环化N1-甲基鸟苷催化第二步形成三环结构。在本研究中，我们在2.4A分辨率下解析了简氏甲烷球菌TYW1的晶体结构。TYW1假定TIM桶具有(/)6拓扑结构，与已报道的自由基SAM酶的结构非常相似。因此，TYW1被认为是通过利用自由基中间体来催化环化反应。通过与其他自由基SAM酶的比较，我们构建了一个由S-腺苷蛋氨酸和两个[4Fe-4S]簇组成的模型结构。酵母中的突变分析支持了这种复杂模型结构的有效性，它提供了对两个[4Fe-4S]簇的自由基反应的结构洞察，以创建一个复杂的三环碱基。较少

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 … More 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