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.

Central European Institute of Technology Masaryk University Kamenice 5 62500 Brno Czech Republic

Department of Molecular Biology Umeå University Building 6K 6L University Hospital Area 90187 Umeå Sweden

Department of Molecular Biosciences The Wenner Gren Institute Stockholm University Stockholm 10691 Sweden

Institute for Biochemistry and Molecular Biology University of Hamburg Martin Luther King Platz 6 20146 Hamburg Germany

Laboratory for Molecular Infection Medicine Sweden Umeå University Building 6K and 6L University Hospital Area 90187 Umeå Sweden

School of Life Science University of Sussex Brighton BN19RH UK

University of Tartu Institute of Technology 50411 Tartu Estonia

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A unified dinucleotide alphabet describing both RNA and DNA structures