Structural insights into assembly of the ribosomal nascent polypeptide exit tunnel

The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.


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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Ning Gao, John L Woolford, Jr.
Aug 20, 2020 Cryo-EM data were recorded using the software Serial EM v3.6, and raw particles were randomly selected using the computer program RELION 3.0; Images for fluorescence microscopy were acquired using ZEN 2 software, blue edition (Zeiss) Cryo-EM data were processed using MotionCor2 v1.1.0, Gctf v1.06, Chimera v1.11.2, Pymol v2.x, Coot v0.8.7 and Phenix v1.18.2; Protein Pilot 5.0 was used to obtain iTRAQ ratios as an average of all peptides for each protein; Images for fluorescence microscopy were processed using Fiji for Mac OSX (National Institutes of Health).
The cryo-EM density maps of the R1, R2 classes of the rpl463-87 mutant particles and the N1, N2, N3, N4 classes of the nog1C rei1C reh1C mutant particles have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-30170, EMD-30174, EMD-30172, EMD-30173, EMD-30175 and EMD-30176, respectively; and the atomic models of the R1 and R2 classes of the rpl463-87 mutant particles have been deposited in the Protein Data Bank (PDB) under accession numbers 7BT6 and 7BTB, respectively. The PDB or EMD files for publicly available cryo-EM structures used in this study are listed as follows: Nsa1 state 2 (6C0F), Nsa1 state C (6EM1), Nsa1 state E (6ELZ), Nog2 state 1 (3JCT), Rix1/Rea1 particle (5FL8), Arx1 particle (5APN), Nmd3 particle (5H4P), and the mature 80S crystal structure of the yeast ribosome (4V88 Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

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All studies must disclose on these points even when the disclosure is negative. For the cryo-EM data, we collected >5,000 raw movie micrographs using a Titan Krios electron microscope. We chose 382,478 raw particles for the rpl463-87 mutant and 332,847 raw particles for the nog1C rei1C reh1C mutant. After 2D classification, all particles for the rpl463-87 mutant were used to produce clear 2D averages retained in the dataset. After 3D classification, the raw particles were applied for high-resolution refinement resulted in the final 3D maps of seven different classes. After 2D classification of the nog1C rei1C reh1C mutant, 297,685 "good" particles that produced clear 2D averages were retained in the dataset. After 3D classification, the raw particles were applied for high-resolution refinement resulted in the final 3D maps of four major classes. The sample size was deemed sufficient because the data yielded our targeted resolution.
Regarding the cryo-EM raw micrograph screening, exclusions were done based on the quality of the images and the presence of ice contamination. Regarding the particle selection, 2D and 3D classification were used, and criterion was based on the quality of resulting 2D class average and 3D maps. This criteria is empirical, but is a standard image processing practice in the cryo-EM community.
For the cryo-EM data, the reproducibility lies in a large number of particles used to derive final 3D maps. Reliability and resolution are measured by gold-standard Fourier shell correlation (Supplementary Figures 2c, 2d, 7c, and 8). The replication efforts through multiple refinement runs were successful and yielded similar 3D maps. For all other experiments, there is a section in the Methods, titled "Statistics and Reproducibility", stating the number of times each experiment was performed for relevant figures. All of our duplicated experiments are biological replicates, not technical replicates.
The raw particles were randomly selected by a computer program (RELION 3.0). Cryo-EM reconstructions use two randomized half-sets to prevent over-refinement of the model, and to assess the resolution of the final model.