Architecture of the yeast Elongator complex

The highly conserved eukaryotic Elongator complex performs specific chemical modifications on wobble base uridines of tRNAs, which are essential for proteome stability and homeostasis. The complex is formed by six individual subunits (Elp1‐6) that are all equally important for its tRNA modification activity. However, its overall architecture and the detailed reaction mechanism remain elusive. Here, we report the structures of the fully assembled yeast Elongator and the Elp123 sub‐complex solved by an integrative structure determination approach showing that two copies of the Elp1, Elp2, and Elp3 subunits form a two‐lobed scaffold, which binds Elp456 asymmetrically. Our topological models are consistent with previous studies on individual subunits and further validated by complementary biochemical analyses. Our study provides a structural framework on how the tRNA modification activity is carried out by Elongator.     

Heterologous expression of dammarenediol synthase gene in an engineered Saccharomyces cerevisiae

Aims: Dammarenediol production by an engineered yeast Saccharomyces cerevisiae was investigated. Methods and Results: A dammarenediol‐producing engineered yeast was constructed by heterologous expression of the dammarenediol synthase gene from Panax ginseng hairy roots through RT‐PCR. Fermentation was carried out in a 5‐L GRJY‐bioreactor with an inoculum size of 1% v/v at 30°C. Dammarenediol detection was performed with silica gel chromatography and HPLC. Determination of dammarenediol synthase activity subcellular distribution was carried out by surveying the enzyme activity in microsomes, lipid particles and total yeast homogenate. When cultured under aerobic conditions, the engineered yeast could produce dammarenediol up to 250 μg l^?1. However, when an anaerobic shift strategy was employed, dammarenediol accumulated at a level as twice as that under aerobic condition. The dammarenediol synthase and dammarenediol were mainly localized in lipid particles. Conclusions: Dammarenediol could be heterologously produced in engineered yeast. The heterologously expressed dammarenediol synthase is mainly localized in lipid particles. Anaerobic shift strategy could enhance the dammarenediol level in the engineered yeast. Significance and Impact of the Study: This study showed that the high‐value plant product dammarenediol could be produced by heterologous expression of the according gene in yeast. Furthermore, the anaerobic shift strategy could be potentially applied in oxidosqualene‐derived compounds production in yeast. Here, the information about subcellular distribution of heterologously expressed dammarenediol synthase in the engineered yeast was also provided.     

Escape of Sgs1 from Rad9 inhibition reduces the requirement for Sae2 and functional MRX in DNA end resection

Homologous recombination requires nucleolytic degradation (resection) of DNA double‐strand break (DSB) ends. In Saccharomyces cerevisiae, the MRX complex and Sae2 are involved in the onset of DSB resection, whereas extensive resection requires Exo1 and the concerted action of Dna2 and Sgs1. Here, we show that the checkpoint protein Rad9 limits the action of Sgs1/Dna2 in DSB resection by inhibiting Sgs1 binding/persistence at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1‐ss mutant variant or by deletion of RAD9, the requirement for Sae2 and functional MRX in DSB resection is reduced. These results provide new insights into how early and long‐range resection is coordinated.