Fig 2: Analysis of in vivo ribosome functional states by ribosome profiling Immunoblot of eEF3 depletion over time. Same amount of cells were harvested at indicated time points, lysed, and subjected to immunoblotting using antibodies against eEF3 or PGK1.Growth of WT and eEF3d cells on YPGR and YPD + auxin plates. Plates were incubated at 30°C for 1 day.Representative run‐off polysome profiles for WT and eEF3d strains. Polysome‐to‐monosome ratios (P/M) are normalized to WT ratios. Data are presented as mean ± SD, n = 2.Polysome profiles from WT or eEF3d cells with CHX added during cell lysis to stop translation (top). Fractions were analyzed by immunoblotting using antibodies against eEF1A, eEF2, or RPL4 (bottom).De‐enriched peptide motifs associated with ribosome pausing at the E, P, and A sites in the absence of eEF3. Peptide motif logos from two biological replicates are shown.

Fig 3: Di- and tripeptide formation ATime courses of MetPhe formation at different eEF3 concentrations. Data are normalized to Met-Phe formation in the presence of 4 µM eEF3 with the maximum value in the dataset set to 1.BMet-Val formation monitored upon rapidly mixing initiation complexes (80S IC; 1 µM) with ternary complexes eEF1A–GTP–[14C]Val-tRNAVal (0.2 µM) in the presence (cyan, 0.78 ± 0.1/s) or absence (orange, 0.34 ± 0.03/s) of eEF3 in a quench-flow apparatus, and the extent of peptide formation was analyzed by HPLC and radioactivity counting. Data are normalized to Met-Val formation in the presence of eEF3 with the maximum value in the dataset set to 1. Data presented as mean ± SEM of n = 3 biological replicates.CMet-Val-Phe formation upon rapid mixing of 80S complexes carrying MetVal-tRNAVal (80S 2C) with ternary complexes eEF1A–GTP–[14C]Phe-tRNAPhe in the presence of eEF2 and eEF3 (green, 0.3 ± 0.02/s), eEF2 (red, 0.03 ± 0.006/s). Data are normalized to Met-Val-Phe formation in the presence of eEF2 and eEF3 with the maximum value in the dataset set to 1. Data presented as mean ± SEM of n = 3 biological replicates.D–FComparison of time courses of 80S 2C reaction with Pmn. 80S 2C complexes with MetPhe-tRNAPhe in the presence of eEF2 and eEF3 (D), eEF2 (E) or eEF3 (F), or in the absence of eEF2 and eEF3 (brown triangles), with Pmn in a quench-flow apparatus. As indicated, the reaction was started either by mixing all components, or by addition of Pmn to a mixture of 80S 2C with the factors preincubated for 15 min. The extent of MetPhe-Pmn formation was analyzed by HPLC and radioactivity counting. Data are normalized to Met-Phe-Pmn formation in the presence of eEF2 and eEF3 (D), eEF2, (E) or eEF3 (F) with the maximum value in the dataset set to 1. Data presented as mean ± SEM of n = 3. For comparison, data from Fig 2B are plotted.GMet-Phe-Val formation upon rapid mixing of 80S complexes carrying either [3H]Met-tRNAi Met (green) or [3H]Met-tRNAfMet(flu) (orange) MetPhe-tRNAPhe (80S 2C) with ternary complexes eEF1A–GTP–[14C]Val-tRNAVal in the presence of eEF2 and eEF3. Data are normalized to Met-Phe-Val formation in the presence of non-labeled initiator tRNA (green) with the maximum value in the dataset set to 1. Source data are available online for this figure.

Fig 4: eEF3 is essential for tri- and polypeptide formation Schematic of the translation elongation cycle as studied here.Met-Phe formation monitored upon rapidly mixing initiation complexes (80S IC) with ternary complexes eEF1A–GTP–[14C]Phe-tRNAPhe in the absence (orange, 0.13 ± 0.02/s) or presence (cyan, 0.15 ± 0.02/s) of eEF3 (2 µM) in a quench-flow apparatus, and the extent of peptide formation was analyzed by HPLC and radioactivity counting. Data are normalized to Met-Phe formation in the absence of eEF3 with the maximum value in the dataset set to 1. Data are presented as mean ± SEM of n = 3 biological replicates.Met-Phe-Val formation monitored upon rapidly mixing 80S complexes carrying MetPhe-tRNAPhe (80S 2C) with ternary complexes eEF1A–GTP–[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green, 0.15 ± 0.01/s), eEF2 (red), or eEF3 (blue). Data presented as mean ± SEM of n = 3 biological replicates.Met-Phe-Val formation monitored with 80S 2C incubated with eEF2 and eEF3 (green, 0.4 ± 0.14/s), eEF2 (red, 1.0 ± 0.5/s), or eEF3 (blue), or in the absence of eEF2 and eEF3 (brown) for 15 min before adding with ternary complexes eEF1A–GTP–[14C]Val-tRNAVal and monitoring the kinetics of Val incorporation. Data presented as mean ± SEM of n = 3. Data information: Datasets in (C) and (D) are normalized to Met-Phe-Val formation in presence of eEF2 and eEF3 with the maximum value set to 1. Source data are available online for this figure.

Fig 5: Rotated Saccharomyces cerevisiae ribosome partially bound by disordered eEF3 ligand ACryo-EM reconstruction of the eEF2 bound 80S ribosome adopting a rotated POST-1 state bearing chimeric ap/P- and pe/E-tRNA as well as a disordered eEF3.BSubunit rotation and head swivel observed in the S. cerevisiae eEF3-80S complex derived from the selected class from the 3D classification shown in (A). 18S rRNA structures illustrating the degree of rotation of the small subunit relative to the large subunit based alignments of the large subunit from a non-rotated structure reference (PDB ID: 6SNT) (Matsuo et al, 2020). The degree of head swiveling (right side) is illustrated based on alignments of the 18S rRNA body from a non-headed swivel reference structure (PDB ID: 6SNT) (Matsuo et al, 2020).C, D80S ribosome in rotated (C) PRE-3 state containing an A/A- and P/E-tRNA and (D) PRE-4 state bearing the hybrid A/P- and P/E-tRNA and bound by the disordered eEF3 ligand.E, FRepresentation of the rotation of the subunit and the 40S-head swivel shown for volumes depicted in (C) and (D), respectively.