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Ribosome-dependent activation of stringent control

Nature volume 534, pages 277–280 (2016)Cite this article

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Abstract

In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation1,2 that leads to rapid and comprehensive reprogramming of metabolic and transcriptional patterns3. In general, transcription of genes for growth and proliferation is downregulated, while those important for survival and virulence are upregulated4. Amino acid starvation is sensed by depletion of the aminoacylated tRNA pools5, and this results in accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA in the ribosomal A site6,7. RelA is recruited to stalled ribosomes and activated to synthesize a hyperphosphorylated guanosine analogue, (p)ppGpp8, which acts as a pleiotropic secondary messenger. However, structural information about how RelA recognizes stalled ribosomes and discriminates against aminoacylated tRNAs is missing. Here we present the cryo-electron microscopy structure of RelA bound to the bacterial ribosome stalled with uncharged tRNA. The structure reveals that RelA utilizes a distinct binding site compared to the translational factors, with a multi-domain architecture that wraps around a highly distorted A-site tRNA. The TGS (ThrRS, GTPase and SpoT) domain of RelA binds the CCA tail to orient the free 3′ hydroxyl group of the terminal adenosine towards a β-strand, such that an aminoacylated tRNA at this position would be sterically precluded. The structure supports a model in which association of RelA with the ribosome suppresses auto-inhibition to activate synthesis of (p)ppGpp and initiate the stringent response. Since stringent control is responsible for the survival of pathogenic bacteria under stress conditions, and contributes to chronic infections and antibiotic tolerance, RelA represents a good target for the development of novel antibacterial therapeutics.

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Figure 1: Structure of RelA bound to the ribosome.
Figure 2: Molecular basis for the recognition of uncharged A-site tRNA.
Figure 3: Interactions between RelA and the ribosome.

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Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The maps have been deposited with the EMDB under accession codes 8107– 8115. Coordinates have been deposited with the Protein Data Bank under the accession code 5IQR.

References

  1. Sands, M. K. & Roberts, R. B. The effects of a tryptophan-histidine deficiency in a mutant of Escherichia coli. J. Bacteriol. 63, 505–511 (1952)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Stent, G. S. & Brenner, S. A genetic locus for the regulation of ribonucleic acid synthesis. Proc. Natl Acad. Sci. USA 47, 2005–2014 (1961)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  3. Hauryliuk, V., Atkinson, G. C., Murakami, K. S., Tenson, T. & Gerdes, K. Recent functional insights into the role of (p)ppGpp in bacterial physiology. Nat. Rev. Microbiol. 13, 298–309 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Magnusson, L. U., Farewell, A. & Nyström, T. ppGpp: a global regulator in Escherichia coli. Trends Microbiol. 13, 236–242 (2005)

    Article  CAS  PubMed  Google Scholar 

  5. Fangman, W. L. & Neidhardt, F. C. Protein and ribonucleic acid synthesis in a mutant of Escherichia coli with an altered aminoacyl ribonucleic acid synthetase. J. Biol. Chem. 239, 1844–1847 (1964)

    Article  CAS  PubMed  Google Scholar 

  6. Haseltine, W. A. & Block, R. Synthesis of guanosine tetra- and pentaphosphate requires the presence of a codon-specific, uncharged transfer ribonucleic acid in the acceptor site of ribosomes. Proc. Natl Acad. Sci. USA 70, 1564–1568 (1973)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. Pedersen, F. S., Lund, E. & Kjeldgaard, N. O. Codon specific, tRNA dependent in vitro synthesis of ppGpp and pppGpp. Nat. New Biol. 243, 13–15 (1973)

    CAS  PubMed  Google Scholar 

  8. Haseltine, W. A., Block, R., Gilbert, W. & Weber, K. MSI and MSII made on ribosome in idling step of protein synthesis. Nature 238, 381–384 (1972)

    Article  ADS  CAS  PubMed  Google Scholar 

  9. Cashel, M. & Gallant, J. Two compounds implicated in the function of the RC gene of Escherichia coli. Nature 221, 838–841 (1969)

    Article  ADS  CAS  PubMed  Google Scholar 

  10. Cashel, M. & Kalbacher, B. The control of ribonucleic acid synthesis in Escherichia coli. V. Characterization of a nucleotide associated with the stringent response. J. Biol. Chem. 245, 2309–2318 (1970)

    Article  CAS  PubMed  Google Scholar 

  11. Atkinson, G. C., Tenson, T. & Hauryliuk, V. The RelA/SpoT homolog (RSH) superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life. PLoS One 6, e23479 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Bai, X.-C., Rajendra, E., Yang, G., Shi, Y. & Scheres, S. H. Sampling the conformational space of the catalytic subunit of human γ-secretase. elife 4, e11182 (2015)

    Article  PubMed  PubMed Central  Google Scholar 

  13. Agirrezabala, X. et al. The ribosome triggers the stringent response by RelA via a highly distorted tRNA. EMBO Rep. 14, 811–816 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Schmeing, T. M. et al. The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA. Science 326, 688–694 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. Selmer, M. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 1935–1942 (2006)

    Article  ADS  CAS  PubMed  Google Scholar 

  16. Lill, R., Robertson, J. M. & Wintermeyer, W. Affinities of tRNA binding sites of ribosomes from Escherichia coli. Biochemistry 25, 3245–3255 (1986)

    Article  CAS  PubMed  Google Scholar 

  17. Schilling-Bartetzko, S., Franceschi, F., Sternbach, H. & Nierhaus, K. H. Apparent association constants of tRNAs for the ribosomal A, P, and E sites. J. Biol. Chem. 267, 4693–4702 (1992)

    Article  CAS  PubMed  Google Scholar 

  18. Sprinzl, M. & Richter, D. Free 3′-OH group of the terminal adenosine of the tRNA molecule is essential for the synthesis in vitro of guanosine tetraphosphate and pentaphosphate in a ribosomal system from Escherichia coli. Eur. J. Biochem. 71, 171–176 (1976)

    Article  CAS  PubMed  Google Scholar 

  19. Friesen, J. D., Fiil, N. P., Parker, J. M. & Haseltine, W. A. A new relaxed mutant of Escherichia coli with an altered 50S ribosomal subunit. Proc. Natl Acad. Sci. USA 71, 3465–3469 (1974)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wendrich, T. M., Blaha, G., Wilson, D. N., Marahiel, M. A. & Nierhaus, K. H. Dissection of the mechanism for the stringent factor RelA. Mol. Cell 10, 779–788 (2002)

    Article  CAS  PubMed  Google Scholar 

  21. Gropp, M., Strausz, Y., Gross, M. & Glaser, G. Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain. J. Bacteriol. 183, 570–579 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Schreiber, G. et al. Overexpression of the relA gene in Escherichia coli. J. Biol. Chem. 266, 3760–3767 (1991)

    Article  CAS  PubMed  Google Scholar 

  23. Valle, M. et al. Locking and unlocking of ribosomal motions. Cell 114, 123–134 (2003)

    Article  CAS  PubMed  Google Scholar 

  24. Maris, C., Dominguez, C. & Allain, F. H. T. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J. 272, 2118–2131 (2005)

    Article  CAS  PubMed  Google Scholar 

  25. Hogg, T., Mechold, U., Malke, H., Cashel, M. & Hilgenfeld, R. Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response. Cell 117, 57–68 (2004)

    Article  CAS  PubMed  Google Scholar 

  26. Yang, X. & Ishiguro, E. E. Dimerization of the RelA protein of Escherichia coli. Biochem. Cell Biol. 79, 729–736 (2001)

    Article  CAS  PubMed  Google Scholar 

  27. English, B. P. et al. Single-molecule investigations of the stringent response machinery in living bacterial cells. Proc. Natl Acad. Sci. USA 108, E365–E373 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Voorhees, R. M. & Ramakrishnan, V. Structural basis of the translational elongation cycle. Annu. Rev. Biochem. 82, 203–236 (2013)

    Article  CAS  PubMed  Google Scholar 

  29. Li, W. et al. Effects of amino acid starvation on RelA diffusive behavior in live Escherichia coli. Mol. Microbiol. 99, 571–585 (2016)

    Article  CAS  PubMed  Google Scholar 

  30. Nguyen, D. et al. Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. Science 334, 982–986 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2015)

    Article  ADS  PubMed  CAS  Google Scholar 

  33. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007)

    Article  CAS  PubMed  Google Scholar 

  34. Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Scheres, S. H. Beam-induced motion correction for sub-megadalton cryo-EM particles. elife 3, e03665 (2014)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003)

    Article  CAS  PubMed  Google Scholar 

  37. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2013)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Noeske, J. et al. High-resolution structure of the Escherichia coli ribosome. Nat. Struct. Mol. Biol. 22, 336–341 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    Article  CAS  PubMed  Google Scholar 

  40. Fischer, N. et al. Structure of the E. coli ribosome-EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM. Nature 520, 567–570 (2015)

    Article  ADS  PubMed  CAS  Google Scholar 

  41. Voorhees, R. M., Schmeing, T. M., Kelley, A. C. & Ramakrishnan, V. The mechanism for activation of GTP hydrolysis on the ribosome. Science 330, 835–838 (2010)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  42. Borovinskaya, M. A. et al. Structural basis for aminoglycoside inhibition of bacterial ribosome recycling. Nat. Struct. Mol. Biol. 14, 727–732 (2007)

    Article  CAS  PubMed  Google Scholar 

  43. Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9, 40 (2008)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D 71, 136–153 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2009)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Amunts, A. et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  47. DeLano, W. L. The PyMOL molecular graphics system. (2002)

  48. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Merritt, E. A. & Bacon, D. J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997)

    Article  CAS  PubMed  Google Scholar 

  50. Stivala, A., Wybrow, M., Wirth, A., Whisstock, J. C. & Stuckey, P. J. Automatic generation of protein structure cartoons with Pro-origami. Bioinformatics 27, 3315–3316 (2011)

    Article  CAS  PubMed  Google Scholar 

  51. Baker, N. A., Sept, D., Joseph, S. & Holst, M. J. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank A. Kelley for providing tRNAs, A. Xu and J. Murray for their contributions to the early stages of this project, C. G. Savva for help with data collection, J. Grimmett and T. Darling for computing support, and X. Bai, G. Murshudov, S. H. W. Scheres, and S. Tan for discussions. This work was supported by grants to V.R. from the UK Medical Research Council (MC_U105184332), the Wellcome Trust (WT096570), the Agouron Institute, and the Louis-Jeantet Foundation.

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Author notes

  1. Israel S. Fernández

    Present address: †Present address: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.,

  2. Alan Brown and Israel S. Fernández: These authors contributed equally to this work

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK

    Alan Brown, Israel S. Fernández, Yuliya Gordiyenko & V. Ramakrishnan

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Contributions

A.B., I.S.F., and V.R. designed the study. Y.G. purified ribosomes. I.S.F. prepared samples, optimized conditions and collected data. A.B. processed data and interpreted the cryo-electron microscopy reconstructions. A.B., I.S.F., and V.R. wrote the manuscript. All authors discussed and commented on the final manuscript.

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Correspondence to V. Ramakrishnan.

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Extended data figures and tables

Extended Data Figure 1 In silico 3D classification scheme.

a, All particles were subjected to 2D classification, from which non-ribosomal particles were discarded, before 3D refinement. To isolate particles containing A-site tRNA and RelA, 3D classification focused on occupancy of the ribosomal A site was performed. Refinement of these 183,615 particles resulted in a reconstruction with a nominal resolution of 2.9 Å. A second round of 3D classification isolated 164,353 well-aligned particles. Conformational heterogeneity of the ribosome was resolved by 3D classification without alignment, which identified two dominant classes in which the body of the small subunit occupies different positions (indicated with an arrow). Class 1 was used as the reference for model building, refinement, and interpretation. To resolve additional conformational heterogeneity of RelA, focused classification with signal subtraction (FCwSS) was performed on each domain, with the hydrolase (HYD) and synthetase (SYN) domains treated as a single unit. For the RRM, zinc-finger and TGS domains a single class was isolated in which the density was better resolved than in the reference class. The overall resolution of the reconstructions are reported according to the Fourier shell correlation (FSC) = 0.143 criterion. Multiple conformations of the HYD and SYN domains were identified, with the four best-resolved classes shown. Together these account for 42% of the particles. b, The two main classes for the TGS domain provide an example of the small conformational differences that were isolated using FCwSS.

Extended Data Figure 2 Quality of maps and models.

a, FSC curve for the EM map. b, The unfiltered and unsharpened density map, in both surface and slice view, coloured by local resolution. c, Fit of models to maps. FSC curves calculated between the refined model and the final map (black), with the self- and cross-validated correlations in blue and magenta, respectively. Information beyond 3.0 Å was not used during refinement and preserved for validation. d, e, Examples of high-resolution features of the map. d, Density for selected rRNA modifications and paromomycin. e, Density for the codon–anticodon interaction in the A site. e, Unfiltered and unsharpened map of RelA bound to the bacterial ribosome, showing the ribosome-binding RelA domains coloured by local resolution according to the FCwSS maps (see Extended Data Fig. 1). The regions amplified in panels f and g are highlighted. f, The RelA ZFD and RRM coloured by local resolution. g, The TGS domain coloured by local resolution.

Extended Data Figure 3 Examples of RelA density.

a, Density for the interaction between the 3′ CCA of the A/T-tRNA and the TGS domain. b, A modelled tRNAAla demonstrates that even the smallest aminoacyl groups would clash with RelA. The sphere size of the atoms of the aminoacyl group corresponds to their van der Waals radii. c, C74 stacks with His432 and C75 can potentially interact with Arg438 of the TGS domain. d, Density for helix α4 of the TGS domain. e, Density for TGS α3. f, Density for the interaction between the ZFD and uS19, showing distinctive density for two consecutive histidine residues. g, Example of side chain density used for the de novo building of the ZFD.

Extended Data Figure 4 RelA domains are connected by flexible linkers.

Two related views showing the density that connects the RelA HYD, SYN and TGS domains with the ZFD/RRM. The linker runs between the A/T-tRNA and the ribosome, but remains flexible as suggested by the weak and broken density. The figure shows the unfiltered, unsharpened density map for the ribosomal small subunit (SSU) of class 4 (Extended Data Fig. 1) with the large subunit removed for clarity.

Extended Data Figure 5 The conformation of uncharged A-site tRNA in the presence of RelA is distinct from aminoacylated A/T-tRNA in the presence of EF-Tu.

a, The ASLs of A-site tRNA (purple) and A/T-tRNA (grey) superpose until base-pair 27:43. At this point, the A-site tRNA is distorted so that the tRNA elbow regions are separated by a 6° rotation. b, A second 11° rotation occurs at base-pair 7:66 of the acceptor stem so that the A-site tRNA in the presence of RelA is closer to the ribosomal SRL.

Extended Data Figure 6 RelA topology diagram.

Secondary structure elements for RelA residues 404–740 are numbered separately for each domain. Unbuilt sections are shown as dashed lines. Topologies were extracted using Pro-origami50.

Extended Data Figure 7 RelA binds RNA through electropositive surfaces.

a, The ZFD and RRM of RelA act together to recognize the ASF of the LSU rRNA. b, As in a, but with the ZFD and RRM shown in surface representation coloured by electrostatic potential. c, The RelA TGS domain binds the acceptor arm of the A/T-tRNA. d, As in c, but with the TGS domain in surface representation coloured by electrostatic potential. Electrostatic potentials were calculated using APBS51, where k is Boltzmann’s constant, T is the temperature of the calculation (310 K) and ec is the charge of an electron.

Extended Data Figure 8 RelA contains an RNA recognition motif (RRM).

a, The RRM from RelA binds the ASF (nucleotides 894–899 shown) through the face of the β-sheet. b, RRMs recognize a wide variety of RNA molecules, but share a common fold and a similar protein–RNA interface, for example in the interaction between PRP24 and U6 small nuclear RNA (PDB accession code 4N0T).

Extended Data Table 1 Refinement and model statistics
Full size table
Extended Data Table 2 Modelled residues
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Brown, A., Fernández, I., Gordiyenko, Y. et al. Ribosome-dependent activation of stringent control. Nature 534, 277–280 (2016). https://doi.org/10.1038/nature17675

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