Biology subjects

Pochopien, A. A.

Publications and source records attributed to Pochopien, A. A..

4 recordsLinked to original sources

bioRxiv2024

The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys> isoacceptors in the P site

The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.

molecular biology↗

bioRxiv2020

Structure of Gcn1 bound to stalled and colliding 80S ribosomes

Protein synthesis is essential to cells and requires a constant supply of nutrients. Amino acid starvation leads to accumulation of uncharged tRNAs that promote ribosomal stalling, which is sensed by the protein kinase Gcn2, together with its effector proteins, Gcn1 and Gcn20. Activation of Gcn2 phosphorylates eIF2, leading to a global repression of translation. Fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, which is a negative regulator of Gcn2. Despite the wealth of biochemical data, structures of Gcn proteins on the ribosome have remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the A-site region of the lead ribosome. The lead ribosome is stalled in a non-rotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, as well as Rbg2/Gir2 located in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings provide a structural basis for Rbg2/Gir2 repression of Gcn2, and also reveal that colliding disomes are the substrate for Gcn1 binding, which has important implications not only for Gcn2-activated stress responses, but also for general ribosome quality control (RQC) pathways.

biochemistry↗

bioRxiv2020

eEF3 promotes late stages of tRNA translocation on the ribosome

SummaryIn addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the A and E sites, its exact mechanism of action is unclear. Here we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl-tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slow-down in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo-EM analysis of ex vivo eEF3-ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non-rotated ribosomal states as well as by opening the L1 stalk to release the E-site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

A role for the Saccharomyces cerevisiae ABCF protein New1 during translation termination

Translation on the ribosome 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 ABCF ATPase, New1, is encoded by a non-essential gene with a cold sensitivity and ribosome assembly defect knock-out phenotype. 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 ribosome assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed a combination of 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.

biochemistry↗