Abstract
Cells express many ribosome-interacting factors whose functions and molecular mechanisms remain unknown. Here, we elucidate the mechanism of a newly characterized regulatory translation factor, energy-dependent translational throttle A (EttA), which is an Escherichia coli representative of the ATP-binding cassette F (ABC-F) protein family. Using cryo-EM, we demonstrate that the ATP-bound form of EttA binds to the ribosomal tRNA-exit site, where it forms bridging interactions between the ribosomal L1 stalk and the tRNA bound in the peptidyl-tRNA–binding site. Using single-molecule fluorescence resonance energy transfer, we show that the ATP-bound form of EttA restricts ribosome and tRNA dynamics required for protein synthesis. This work represents the first example, to our knowledge, in which the detailed molecular mechanism of any ABC-F family protein has been determined and establishes a framework for elucidating the mechanisms of other regulatory translation factors.
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Acknowledgements
This work was supported by Howard Hughes Medical Institute and US National Institutes of Health (NIH) grants (R01 GM29169 and GM55440) to J.F.; an NIH grant (2U54 GM074958) and a US National Science Foundation (NSF) grant (0424043) to J.F.H.; a Burroughs Wellcome Fund Career Awards in the Biomedical Sciences (CABS 1004856), an NSF CAREER Award (MCB 0644262) and an NIH National Institute of General Medical Sciences grant (R01 GM084288) to R.L.G. The authors thank R.A. Grassucci for assistance with cryo-EM data collection and M. Thomas and C. Kinz-Thompson for assistance with the preparation of illustrations. We thank members of the Frank, Hunt and Gonzalez laboratories for advice and technical assistance.
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G.B., B.C., J. Fei, J.F.H., R.L.G. and J. Frank designed the experiments. G.B. performed biochemical studies and prepared the biological samples for cryo-EM. B.C. collected cryo-EM data and performed 3D reconstruction. Y.H., B.C. and C.W. performed the modeling, fitting and analysis. W.N. and J. Fei collected and analyzed smFRET data. B.C., G.B., Y.H., W.N., R.L.G., J.F.H. and J. Frank wrote the manuscript; all authors approved the final manuscript.
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Supplementary Figure 1 In vivo pulldown of His6-EttA-EQ2 with 70S ribosome.
Strain MG1655-ettA::Tn5 carrying either the plasmids, pBAD-ettA, pBAD-His6-ettA or pBAD-His6-ettA-EQ21, were grown and protein over-expression was induced. After cells collection and lysis, an equal amount of total protein for each sample was loaded on three columns of Ni-NTA. The columns were washed and EttA was eluted into fractions. (a) Coomassie colored gel of the fractions for the different samples. From this gel some of the bands that specifically appeared in the pBAD-His6-ettA-EQ2 sample compared to the pBAD-His6-ettA (if an equivalent was present in the control pBAD-ettA the band was also selected) were identified by peptide mass fingerprinting identification. These bands are labeled with red numbers. The identification of the protein(s) present in those bands is shown on the table (right side). The table shows the protein ID (Uniprot ID), the pValue, Z-score and Score calculated by Aldente, the predicted molecular weight (Mw), the number of peptides detected for each identification (Hits), the percentage of coverage of the peptides identified against the full protein (Coverage). (b) 5 liters culture of the strain MG1655-ettA::Tn5 carrying pBAD-His6-ettA-EQ2 had been processed like the one presented in (a). The fractions 1 to 5 after Ni-NTA pull-down were pooled, concentrated and run on a gel filtration column. A fast running peak with a high absorbance at 280 nm was detected in the void volume of the column, which corresponded to a molecular weight higher than 1.5 MDa. The fractions of this peak were pooled and concentrated. (c) This sample was separated on an SDS-PAGE gel that shows a full ribosome proteins profile. The sample also had been submitted to western-blot analysis with an affinity purified anti-EttA antibody to confirm the presence of EttA on this purified complex.
Supplementary Figure 2 Peptide formation assay in parallel with cryo-EM sample preparation.
(a) 70S–mRNA–[35S]fMet-tRNAfMet initiation complex (0.5 μM), prepared in parallel with the 70S initiation complex used in cryo-EM study, was first mixed with EttA-EQ2 (6 μM) or polymix buffer and incubated at 37 °C for 1 min, then mixed with Phe-tRNAPhe–EF-Tu–GTP and Lys-tRNALys–EF-Tu–GTP ternary complexes (0.67 μM each) and incubated at 37 °C for 1 min, then with EF-G (1.5 μM) and incubated at 37 °C for indicated reaction time (Supplementary Methods). (b) Relative fractions of mono- (fM), di- (fM-F), and tri- (fM-F-K) peptides in (a) after 1 min incubation with EF-G.
Supplementary Figure 3 Flow chart of RELION-based hierarchical classification and Fourier shell correlation (FSC) curves for reconstructions from the three classes.
(a) RELION-based classification diagram. See computational classification of Supplementary Note for details. (b) FSC curve to determine the resolution of the reconstructions from the three classes. For each class, the two half-volumes were generated using RELION. The FSC between the two half-volumes was calculated using SPIDER (Online Methods). The resolution of each map was determined using FSC = 0.143 cutoff, yielding 7.5 Å (70S–EttA–tRNA2, Class I), 9.1 Å (70S–EttA–tRNA, Class II) and 7.7 Å (70S–EttA, Class III), respectively.
Supplementary Figure 4 R.m.s. deviation (r.m.s.d.) and cross-correlation coefficient (CCC) plots of the MDFF process.
(a) CCC plotted versus the simulation time in picoseconds (ps). (b) RMSD plotted versus the simulation time in picoseconds. In both (a) and (b), the MDFF equilibration phase (left plot) is distinct from the production phase (right plot). Dotted red lines separate consecutive stages of the equilibration phase conducted at decreasing positional harmonic constraints (in kcal/(mol*Å) at the top of the plots). The solid red line denotes the frame at which the convergence is considered achieved as it marks the beginning of a plateau.
Supplementary Figure 5 Significant Pfam-A matches of PtIM of EttA with PF12848 (ABC_tran_2) domain and sequence alignment of E. coli K12-MG1655 tRNA-encoding genes.
(a) HMM, hidden Markov model sequence; MATCH, the match between HMM and SEQ; PP, posterior probability, or the degree of confidence in each individual aligned residue; SEQ, query sequence. Green and cyan highlights residues in PtIM of EttA that may interact with P-site tRNAfMet and with 23S rRNA, respectively (Supplementary Table 1). (b) An alignment of all the genes coding for tRNA in E. coli MG1655 was generated using the tRNAdb3 database (http://trna.bioinf.uni-leipzig.de), highlighting the specific features of the initiator tRNA which may interact with ribosome-bound EttA-EQ2. tRNA structural features that are close to EttA-EQ2 are highlighted in yellow. The CpU bulge, present in two isoforms of Initiator tRNA (Ini) and two of three isoforms of Proline tRNA (Pro), is in green. The C1::A72 mismatch, only present in the two isoforms of initiator tRNA, is in purple. The anticodons of all the tRNAs are in red.
Supplementary Figure 6 Cryo-EM 3D reconstruction of the class III 70S–EttA complex.
Color scheme and abbreviations of landmarks are the same as Fig. 1. Circled region indicates the poorly defined distal end of the EttA PtIM.
Supplementary Figure 7 EttA-mediated regulation of MS-I↔MS-II equilibrium as observed using smFRETL1-tRNA studies of a PRE–APhe complex.
(a) Cartoon diagram of the conformational equilibrium of the PRE-APhe complex between the MS-I conformation harboring an open L1 stalk (ribosomal complex on left) and the MS-II conformation harboring a closed L1 stalk (ribosomal complex on right). The 30S subunit is shown as a tan cartoon, 50S subunit as a blue cartoon; tRNAPhe as an orange ribbon; mRNA as a black curve; Cy3 FRET donor fluorophore as a green circle; and Cy5 FRET acceptor fluorophore as a red circle. (b-d) smFRETL1-tRNA experiments were recorded in the presence of 0.8 mM ATP and in (b) the absence of EttA, (c) the presence of 1.8 μM EttA, and (d) the presence of 1.8 μM EttA-EQ2. 1st row: Representative Cy3 and Cy5 fluorescence intensity (Flour. Int.) vs. time trajectories. The fluorescence intensities are plotted in arbitrary units (a.u.) with the Cy3 fluorescence intensity plotted in green and the Cy5 fluorescence intensity plotted in red. 2nd row: The corresponding EFRET vs. time trajectories. The EFRET at each time point was calculated using EFRET = ICy5 / (ICy3 + ICy5), where ICy3 and ICy5 are emission intensities of Cy3 and Cy5, respectively, and is plotted in blue. 3rd row: Surface contour plots of the time evolution of the population FRET. The contour plots were generated by superimposing individual EFRET vs. time trajectories and the contours are colored from white (lowest-populated) to red (highest-populated). N denotes the number of EFRET vs. time trajectories that were used to construct each contour plot.
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Chen, B., Boël, G., Hashem, Y. et al. EttA regulates translation by binding the ribosomal E site and restricting ribosome-tRNA dynamics. Nat Struct Mol Biol 21, 152–159 (2014). https://doi.org/10.1038/nsmb.2741
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DOI: https://doi.org/10.1038/nsmb.2741
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