A role for the Saccharomyces cerevisiae ABCF protein New1 in translation termination/recycling.

Villu Kasari,Vasili Hauryliuk,Gemma C Atkinson,Yang Zhou,Agnieszka A Pochopien,Jiří Nováček,Marcus J O Johansson,Kathryn Turnbull,Tõnu Margus,Victoriia Murina,Daniel N Wilson,Tracy Nissan,Michael Graf

doi:10.1093/nar/gkz600

Abstract

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3′-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling.

Highlights

NEW1, at both 20◦C and 30◦C (Figure 1B and Supplementary Figure S4BC). While these results are consistent with overlapping functions of New1 and eEF3 in protein synthesis, the additive negative effects on fitness could be due to the two proteins acting on two sequential steps in gene expression, for example, ribosome assembly (New1) and translation
A homologue of New1 and the bacterial ABCF translation factor EttA [55]––Uup––presents an analogous case: while overexpression of Uup supresses both cold sensitivity and ribosome assembly in E. coli caused by knockout of the translational GTPase BipA [14], it is unclear if Uup is an assembly factor or translation factor
Cryo-EM and Ribo-Seq results collectively argue that New1, acts on mature ribosomes, and its loss affects translation termination/recycling, leading to ribosomal pile up in front of stop codons preceded by a lysine or arginine codon

Summary

Introduction

Loss of New results in a ribosome assembly defect in S. cerevisiae and causes cold sensitivity, that is, a growth defect at low temperatures [23]. It is unclear whether the effect is direct, through participation of New in ribosome assembly, or indirect, as perturbation of translation can cause ribosome assembly defects [27]. The latter possibility is supported by the detection of New in polysomal fractions [23], motivating our current investigation.

Methods

Results

Conclusion