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Late steps in bacterial translation initiation visualized using time-resolved cryo-EM

Nature volume 570, pages 400–404 (2019)Cite this article

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Abstract

The initiation of bacterial translation involves the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex to form a 70S initiation complex, which subsequently matures into a 70S elongation-competent complex. Rapid and accurate formation of the 70S initiation complex is promoted by initiation factors, which must dissociate from the 30S initiation complex before the resulting 70S elongation-competent complex can begin the elongation of translation1. Although comparisons of the structures of the 30S2,3,4,5 and 70S4,6,7,8 initiation complexes have revealed that the ribosome, initiation factors and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during formation of the 70S initiation complex, the structures of any intermediates formed during these rearrangements, and the contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, the absence of a structure of the 70S elongation-competent complex formed via an initiation-factor-catalysed reaction has precluded an understanding of the rearrangements to the ribosome, initiation factors and fMet-tRNAfMet that occur during maturation of a 70S initiation complex into a 70S elongation-competent complex. Here, using time-resolved cryogenic electron microscopy9, we report the near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, initiation factor dissociation and fMet-tRNAfMet positioning during formation of the 70S elongation-competent complex. Our results demonstrate the power of time-resolved cryogenic electron microscopy to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.

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Fig. 1: Structural and time-resolved population analyses of the 50S subunit, 70S IC and 70S EC.
Fig. 2: Ribosome, initiation factor and fMet-tRNAfMet dynamics during 70S IC formation.
Fig. 3: Ribosome and fMet-tRNAfMet dynamics during maturation of the 70S IC into a 70S EC.
Fig. 4: Structure-based kinetic model for late steps in bacterial translation initiation.

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Data availability

The cryo-EM reconstruction maps have been deposited in the Electron Microscopy Data Bank (EMDB) server under the accession codes EMD-0643 (30S IC), EMD-0662 (70S IC) and EMD-0661 (70S EC). The structural models obtained by MDFF have been deposited in the Protein Data Bank (PDB) server under accession codes 6O7K (30S IC) and 6O9K (70S IC). The structural model obtained by rigid-body fitting has been deposited in the PDB server under accession code 6O9J (70S EC).

Code availability

A pseudocode describing the control actions of the software synchronizing time-resolved cryo-EM apparatus is available upon request.

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Acknowledgements

This work was supported by funds to J.F. from the National Institutes of Health (R01 GM 55440 and GM 29169) and to R.L.G. from the National Institutes of Health (R01 GM 084288). K.C. was supported by an American Cancer Society Postdoctoral Fellowship (125201).

Reviewer information

Nature thanks A. Amunts, Simpson Joseph and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Sandip Kaledhonkar, Ziao Fu, Kelvin Caban

Authors and Affiliations

  1. Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY, USA

    Sandip Kaledhonkar, Wen Li, Bo Chen & Joachim Frank

  2. Integrated Program in Cellular, Molecular and Biophysical Studies, Columbia University, College of Physicians and Surgeons, New York, NY, USA

    Ziao Fu

  3. Department of Chemistry, Columbia University, New York, NY, USA

    Kelvin Caban & Ruben L. Gonzalez Jr

  4. Department of Biological Sciences, Columbia University, New York, NY, USA

    Ming Sun & Joachim Frank

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  1. Sandip Kaledhonkar
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Contributions

S.K., Z.F., K.C., B.C., M.S., R.L.G. and J.F. designed the research; K.C. prepared all of the biological reagents; S.K. and Z.F. performed the time-resolved cryo-EM experiments; S.K., Z.F. and W.L. analysed the data; S.K., Z.F., K.C., R.L.G. and J.F. wrote the manuscript; all eight authors approved the final manuscript.

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Correspondence to Ruben L. Gonzalez Jr or Joachim Frank.

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Extended data figures and tables

Extended Data Fig. 1 Cryo-EM reconstructions.

a, b, 3D cryo-EM-derived Coulomb potential maps30 of the 30S IC (a) and the 30S subunit + fMet-tRNAfMet complex (b) obtained from a control experiment in which the 30S IC in Tris-polymix buffer and a solution of Tris-polymix buffer lacking 50S subunits were injected into the microfluidic chip designed to give the longest reaction time (~600 ms), mixed, allowed to react, and sprayed onto an electron microscopy grid that was rapidly plunged into liquid ethane. The sizes of the resulting populations of the 30S IC and the 30S subunit + fMet-tRNAfMet complex were 75% and 25%, respectively, which demonstrates that most of the 30S ICs remain intact during the mixing-spraying process. c, Plot of the concentrations of the 50S subunit, 70S IC and 70S EC as a function of time generated by using the initial 50S subunit and 30S IC concentrations analogous to those used in our mixing-spraying microfluidic chip (that is, 0.6 µM and 1.2 µM, respectively) and modelling the kinetics of subunit joining using the kinetic scheme and set of rate constants reported previously for a subunit-joining reaction performed in the presence of IF1 and IF2, but in the absence of the IF319. A detailed description of the kinetic modelling can be found in the Methods. The plot predicts that the 70S IC population should peak within 50–250 ms after mixing of the 50S subunit and 30S IC, and that these 70S ICs should mature to a notable population of 70S ECs within the next several hundreds of milliseconds. Therefore, to ensure that we would capture formation of the 70S IC and its maturation to the 70S EC, we selected microfluidic chips designed to provide reaction times of approximately 20 ms, 80 ms, 200 ms and 600 ms. The free 50S subunit, 70S IC and 70S EC populations observed in our time-resolved cryo-EM experiments are shown as blue diamonds, light grey circles and dark grey triangles, respectively.

Extended Data Fig. 2 Time-resolved cryo-EM grid preparation apparatus.

a, A photograph of the mixing-spraying, time-resolved cryo-EM apparatus, labelled to show all major components. The mixing-spraying microfluidic chip is mounted inside an environmentally controlled chamber. A syringe pump, which is controlled by a laboratory-written, Visual Basic and C++ software program called Howard5e45, is used to inject the reactants from inlets 1 and 2 into the microfluidic chip. Once in the microfluidic chip, the reactants are mixed and allowed to react for the reaction time specific to the microfluidic chip being used. The electron microscopy grid is held at the end of the plunger by a pair of tweezers. The Howard5e software controls and synchronizes the syringe pump as well as the plunger that holds the tweezer-mounted electron microscopy grid45. Thus, as the sprayers discharge the reaction from the microfluidic chip onto the electron microscopy grid, the plunger is activated to plunge the grid into cryogen45. b–f, Images of cryo-EM grids prepared by the mixing-spraying, time-resolved cryo-EM apparatus, going from low to high magnification. b, Grid-view depicting droplets of different sizes deposited on the grid. c, Square-view depicting droplet distribution over the holes. d, Hole-view depicting ice distribution over holes. For image acquisition, thin ice regions were selected. e, A representative micrograph showing good particle density. f, Power spectrum of the acquisition image in e.

Source Data

Extended Data Fig. 3 Flow-chart of the work process for single-particle analysis and 3D refinement.

In the first step, particles were auto-picked from the images recorded for the individual time points. Auto-picked particles were then extracted using 2× binning of the images and subjected to 2D classification to discard ice-like and/or debris-like particles and define 30S subunit-like, 50S subunit-like and 70S ribosome-like particle classes. Representative 2D classes of 30S subunit-like, 50S subunit-like and 70S ribosome-like particles are shown on the left and right of the flow chart. A detailed account of the classification scheme is provided in the Methods. In brief, following 2D classification at each time point, two particle datasets were created. The first particle dataset was composed of 170,864 30S subunit-like projections, and the second was composed of 144,504 50S subunit-like and 70S ribosome-like projections. The first particle dataset with 170,864 30S subunit-like projection classes was subjected to 3D classification, which yielded two major subclasses. The first of these contained 86,367 30S ICs and the second contained 17,686 30S subunits carrying only fMet-tRNAfMet (that is, 30S subunit + fMet-tRNAfMet complexes). The second particle dataset, containing the 144,504 50S subunit-like and 70S ribosome-like particles, was also subjected to a combination of 3D and 2D classification to separate compositional heterogeneity consisting of the 50S subunit ribosome and 70S ribosome. After performing a combination of 3D and 2D classifications, two particle datasets were created, the first containing 50,918 50S subunit particles and the second containing 80,138 70S ribosome-like particles. Further 3D classification was performed on the dataset containing 80,138 70S ribosome-like particles, which yielded 70S IC and 70S EC classes. Particles from 50S subunit, 70S IC and 70S EC were traced back to each time point, as tabulated at the bottom of the flow chart.

Extended Data Fig. 4 Masked classification scheme to look for rare conformations of the 70S IC and 70S EC.

a–c, The mask (grey) covering densities of IF1 (magenta), IF2 (purple), P/P-tRNA (orange) and P/E-tRNA is shown in different views. The views depict the position of the mask with respect to the 30S subunit (pale yellow) and the 50S subunit (blue). d, For the masked 3D classification scheme, this mask was applied to the dataset of refined 80,138 70S particles, which yielded mostly three types of class. The first class encompasses 44% of the particles with density for IF2 (purple) and tRNA in the P/I position (green), the second class encompasses 48% with density for tRNA in the P/P position (orange), and the third class encompasses approximately 8% without any density in the masked region. The 3D refinement of the first, second and third types of class yielded cryo-EM maps of 70S IC, 70S EC and low-resolution 70S EC, respectively.

Extended Data Fig. 5 Selection of noise particles and angular coverage of 70S IC with addition of noisy particles.

a, Noise particles selected from the gain-corrected micrograph. The noise particles that did not exhibit any ribosome particle-like features were selected from the background (green circles). b, The angular coverage of the 70S IC with respect to the view depicted in the centre panel, as a function of the level of added noise.

Extended Data Fig. 6 Fourier shell curves and cryo-EM reconstructions for the 30S IC, 70S IC and 70S EC.

a–c, Fourier shell curves (FSC) for the 30S IC (a), 70S IC (b) and 70S EC (c). The resolutions of these structures were estimated using a resolution-estimating protocol that avoids overfitting and uses the FSC and 0.143 criterion28. d–f, Cryo-EM reconstructions of 30S IC (d), 70S IC (e) and 70S EC (f). g–i, Angular orientation coverage of 30S IC (g), 70S IC (h), and 70S EC (i), presented corresponding to the views depicted in d, e and f, respectively. j–l, Directional FSC plots46 of the cryo-EM reconstructions of the 30S IC (j), 70S IC (k) and 70S EC (l).

Extended Data Fig. 7 IF2–70S ribosome interactions in the 70S IC.

a, The positions of IF2 (dark purple), 70S P/I fMet-tRNAfMet (green), uS12 (yellow), and H69, H71, H80, H89 and H95 (SRL; all shown in blue) within the cryo-EM reconstruction of the 70S IC (transparent grey) were obtained using the MDFF method. b, A magnified view of the structure shown in a, highlighting the interactions that IF2 makes with the 70S ribosome in the 70S IC.

Extended Data Fig. 8 Portion of the Coulomb potential map corresponding to the guanosine nucleotide obtained from the 4 Å resolution, cryo-EM reconstruction of the 70S IC.

a, b, Rigid-body fitting was used to position either GTP (a) or GDP·Pi (b) into the Coulomb potential map. The Coulomb potential map is shown as a blue mesh. The initial position of Pi relative to GDP for the rigid-body fitting was taken from the structure of the GDP·Pi-form of the G protein Giα1 (PDB code 1AS2)47.

Source Data

Extended Data Fig. 9 Views of the major components of the 30S IC and 70S IC after structural modelling of the 30S IC and 70S IC using the MDFF method.

a, fMet-tRNAfMet (orange) in its 30S P/I configuration in the 30S IC. b, IF1 (magenta) in the 30S IC. c, IF2 (light purple) in the 30S IC. d, fMet-tRNAfMet (green) in its 70S P/I configuration in the 70S IC. e, IF2 (dark purple) in the 70S IC. For each component, the reconstructed Coulomb potential map is represented by the mesh and the structural model is represented by secondary structure cartoons.

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Kaledhonkar, S., Fu, Z., Caban, K. et al. Late steps in bacterial translation initiation visualized using time-resolved cryo-EM. Nature 570, 400–404 (2019). https://doi.org/10.1038/s41586-019-1249-5

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