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

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.

RiboDiPA: a novel tool for differential pattern analysis in Ribo-seq data

“…To extend our single codon analysis, we also applied the RiboDiPA pipeline to data collected from yeast lacking New1, a distinct translation termination regulator, which was published by Kasari et al. ( 63 ). NEW1 is a non-essential gene with a cold sensitivity knockout phenotype.…”

“… RiboDiPA for single-codon resolution DP analysis: NEW1 deletion ( A ) Single-codon DP analysis of data from ( 63 ), comparing wild type yeast to a NEW1 deletion strain at 20°C. Counts of genes that show DP at a particular codon near the start or stop codon are plotted, with genes with a positive log fold change in the new1Δ condition relative to the WT condition in red, and negative log fold change in cyan.…”

“…The second data set was also collected in yeast by Kasari et al. ( 63 ) and compared wild type yeast cells to yeast cells mutant for NEW1 ( new1Δ ), which is an non-essential translation termination factor with a cold sensitivity phenotype. Data was collected at both 30°C and 20°C, but all comparisons in this paper are for 20°C.…”

What monomeric nucleotide binding domains can teach us about dimeric ABC proteins

“…Here, the NBD–NBD association appears to be noncanonical in the apo state when dissociated from the ribosome [79]. A typical NBD–NBD sandwich dimer with nucleotide bound is found in the cryo‐EM structure of ribosome‐bound eEF3, implying a major rotation and rearrangement of the NBDs upon ribosome association [79,80]. The linker between the first and second NBDs in eEF3 is similar to the ABCE‐type linker.…”

CTELS: A Cell-Free System for the Analysis of Translation Termination Rate

“…Such approaches are very convenient for analyzing the efficiency of read-through events, but tell nothing about the kinetics and molecular mechanics of the process. Ribosome profiling was recently applied to study termination and stop codon suppression [48,[69][70][71][72][73] and revealed pervasive ribosome occupancy of mRNA regions downstream of stop codons, likely due to defective or regulated ribosome recycling, reinitiation, mRNA degradation, or other processes [74][75][76][77][78][79][80][81]. However, it is also hardly used for analysis of translation dynamics.…”