Abstract
Faulty or damaged messenger RNAs are detected by the cell when translating ribosomes stall during elongation and trigger pathways of mRNA decay, nascent protein degradation and ribosome recycling. The most common mRNA defect in eukaryotes is probably inappropriate polyadenylation at near-cognate sites within the coding region. How ribosomes stall selectively when they encounter poly(A) is unclear. Here, we use biochemical and structural approaches in mammalian systems to show that poly-lysine, encoded by poly(A), favors a peptidyl-transfer RNA conformation suboptimal for peptide bond formation. This conformation partially slows elongation, permitting poly(A) mRNA in the ribosome’s decoding center to adopt a ribosomal RNA-stabilized single-stranded helix. The reconfigured decoding center clashes with incoming aminoacyl-tRNA, thereby precluding elongation. Thus, coincidence detection of poly-lysine in the exit tunnel and poly(A) in the decoding center allows ribosomes to detect aberrant mRNAs selectively, stall elongation and trigger downstream quality control pathways essential for cellular homeostasis.
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Data availability
The poly(A)-stalled ribosome map has been deposited to the EMDB with accession code EMD-10181. Atomic coordinates have been deposited to the Protein Data Bank under accession code PDB 6SGC. A re-refined version of 5LZV that includes the ester bond between the P-site tRNA and the attached Valine of the nascent chain with the correct bond length (used in Extended Data Fig. 7b,c) is available upon reasonable request. Source data for Figs. 1a, 5a and 6c and Extended Data Fig. 1a,b are available with the paper online. All other data are available upon reasonable request.
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Acknowledgements
We thank T. Tang and L. Passmore for useful discussions, help with circular dichroism measurements and sharing data before publication; J. Grimmett and T. Darling for advice, data storage and high-performance computing; M. Daly, G. Cannone and J. Brown for technical support; S. Scheres, T. Nakane and P. Emsley for advice; the MRC Laboratory of Molecular Biology Electron Microscopy Facility for access and support of electron microscopy, sample preparation and data collection; Diamond for access and support of the Cryo-EM facilities at the UK national Electron Bio-Imaging Centre (eBIC) (proposal no. EM17434-53, funded by the Wellcome Trust, MRC and BBSRC); Z. Yang for data collection support at eBIC; and Hegde and Ramakrishnan laboratory members for useful discussions. This work was supported by the UK Medical Research Council (grant no. MC_UP_A022_1007 to R.S.H. and grant no. MC_U105184332 to V.R.); a Wellcome Trust Senior Investigator award (grant no. WT096570), the Agouron Institute and the Louis-Jeantet Foundation (to V.R.); a Bio-X graduate fellowship (to J.C.); and the NIH (grant nos. GM51266 and GM113078 to J.D.P.).
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V.C. generated the cryo-EM structures, built and interpreted molecular models and wrote the first draft of the manuscript. S.J. prepared and characterized samples for structure determination, performed biochemical and cell assays of stalling and interpreted these data. J.C. and J.D.P. provided supporting data that corroborated the stalling model. A.B. and S.S. produced an initial stalled ribosome structure that seeded the project. V.R. provided overall project guidance and helped interpret the structure. R.S.H. conceived the project, provided overall project guidance, helped interpret the findings and wrote later drafts of the manuscript. All authors contributed to manuscript editing.
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Extended data
Extended Data Fig. 1 Additional characterization of ribosome stalling in vitro.
a, A second example of nascent chain products resulting from in vitro translation of iterated AAG or AAA lysine codons in human cell lysate, as in Fig. 1a. The positions of nascent chain products containing 4, 9, or 12 lysine residues are indicated. b, Analysis of iterated AAG versus AAA codons for stalling in rabbit reticulocyte lysate. The translation reaction was performed for 20 min after which the proportion of stalled products was assessed by the relative amounts of peptidyl-tRNA versus full length polypeptide. The ‘background’ of ~ 20% peptidyl-tRNA even in the absence of stalling is due to failed termination at the stop codon, which is located within a few nucleotides of the 3’ end of the mRNA. Later in vitro stalling experiments with a longer 3’UTR that protrudes outside the mRNA channel showed improved termination efficiency (~ 95%). An overly short 3’UTR presumably makes the mRNA more flexible in the mRNA channel and less able to recruit eRF1. Multiple experiments such as this one were quantified to produce the graph shown in Fig. 1b. c, Time course of the appearance of full length (FL) product for constructs containing the indicated number of iterated AAG or AAA codons. Translation was synchronized by first pausing the ribosome at a run of rare leucine codons just preceding the poly-basic encoding sequence, then restarting translation at time 0 by addition of tRNA. The mean ± SEM for each time point calculated from two experiments are plotted.
Extended Data Fig. 2 Cryo-EM analysis of ribosomes stalled on poly(A).
a, Representative micrograph of poly(A)-stalled ribosomes used for single particle analysis. Scale bar is 50 nm. b, Data processing scheme used for structure determination in Relion 3.0. 3D classification reveals that ~90% of active ribosomes are in the canonical state with P/P tRNA while ~10% are seen in the rotated state with A/P and P/E hybrid state tRNAs. The majority of the rotated state ribosomes also contain density for a preceding ribosome and therefore represent ribosomes that have collided with a poly(A)-stalled ribosome. c, Fourier shell correlation (FSC) curve of the final map illustrating an overall resolution of 2.8 Å.
Extended Data Fig. 3 Characterization of cryo-EM map.
a, Local resolution of the poly(A)-stalled ribosome sliced through the center. The positions of key elements are indicated. PTC: peptidyl-transferase center. Inset (right) highlights the high local resolution at the PTC and decoding center. b, Slices through the density map at the plane of the polypeptide exit tunnel (left) and mRNA channel (right). Continuous nascent chain density corresponding to a mixture of poly-Lys lengths and Cα positions is contoured at a different level to the rest of the map and is shown in magenta, and mRNA density is shown in red. The P site tRNA is green, 40 S subunit in yellow, and 60 S subunit in light blue.
Extended Data Fig. 4 Experimental EM density for P-site Lys-tRNALys,3.
Map-to-model fits for the P-site Lys-tRNA(lys,3) with the AAA codon of the mRNA in the P site and the first amino acid (lysine) of the nascent polypeptide. Base modifications at positions 34 and 37 of the tRNA are shown within the cryo-EM density.
Extended Data Fig. 5 Views of the mRNA density in the EM map of the poly(A)-stalled ribosome.
The density map is sliced through the ribosome in a plane that reveals the decoding center and shows the mRNA within the small subunit. The large and small subunits (blue and yellow, respectively), P-site tRNA (green) and mRNA (red) are colored. The inset shows a zoomed in region of the mRNA channel, illustrating that the poly(A) mRNA is ordered through most of the channel. The bottom panel shows the mRNA density in the P- and A-sites in the final refined and sharpened map. The mRNA is well ordered in the P-site due to base-pairing with the P-site tRNA, and ordered in the A-site due to stabilizing interactions with rRNA as shown in Fig. 3.
Extended Data Fig. 6 Guanosine interrupts the intrinsic helical propensity of poly(A).
Circular dichroism (CD) spectra of AAAAAA (red), AAGAAG (blue) and AAGGAA (green) RNA oligonucleotides are plotted. These spectra are averaged from 9 independent measurements performed on the same samples. The AAAAAA oligo displays a CD signature characteristic for the helical conformation of poly(A), as described previously 52. Introduction of guanosines significantly disrupts this helical structure.
Extended Data Fig. 7 Comparison of peptidyl-tRNA geometry in different mammalian RNC structures.
Shown are the EM density maps for the peptidyl-tRNA region at the PTC for the indicated structures. The fitted models are shown for the poly(A)-stalled ribosome and the RNC stalled at the stop codon with a dominant-negative eRF1AAQ mutant (PDB code 5LZV). The 5LZV RNC is in a geometry competent for peptidyl-transfer (or in this case, peptide release by eRF1). The structure from the didemnin-B stalled RNCs contains a mixture of nascent chains stalled at different positions. Thus, the nascent chain density represents an average of a variety of peptidyl-tRNAs. Note that the nascent chain model from 5LZV fits well into the density map, indicating that the majority of peptidyl-tRNAs assume this configuration during active elongation. The geometry for the poly(A) peptidyl-tRNA is unambiguously different from this optimal geometry. Lys and Val refer to the lysine and valine side chains of modeled nascent chains. The asterisks indicate density for side chains that are not shown.
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Source data
Source Data Fig. 1
Full uncropped gel for Fig. 1a
Source Data Fig. 5
Full uncropped gel for Fig. 5b
Source Data Fig. 6
Example of raw flow cytometry data for Fig. 6c
Source Data Extended Data Fig. 1
Full uncropped gels for Extended Data Fig. 1a and 1b
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Chandrasekaran, V., Juszkiewicz, S., Choi, J. et al. Mechanism of ribosome stalling during translation of a poly(A) tail. Nat Struct Mol Biol 26, 1132–1140 (2019). https://doi.org/10.1038/s41594-019-0331-x
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DOI: https://doi.org/10.1038/s41594-019-0331-x
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