Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3′ of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3′ pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (RelTt). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of RelTt (RelTtNTD) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.
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All the structures have been deposited in the PDB database with the following accession numbers; 6S2V, 6S2T and 6S2U. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Methods. Additional data related to this paper may be requested from the authors.
Laffler, T. & Gallant, J. A. Stringent control of protein synthesis in E. coli. Cell 3, 47–49 (1974).
Cashel, M. & Gallant, J. Two compounds implicated in the function of the RC gene of Escherichia coli. Nature 221, 838–841 (1969).
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).
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).
Stent, G. S. & Brenner, S. A genetic locus for the regulation of ribonucleic acid synthesis. Proc. Natl Acad. Sci. USA 47, 2005–2014 (1961).
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 [corrected]. Cell 117, 57–68 (2004).
Avarbock, D., Salem, J., Li, L. S., Wang, Z. M. & Rubin, H. Cloning and characterization of a bifunctional RelA/SpoT homologue from Mycobacterium tuberculosis. Gene 233, 261–269 (1999).
Mechold, U., Murphy, H., Brown, L. & Cashel, M. Intramolecular regulation of the opposing (p)ppGpp catalytic activities of Rel(Seq), the Rel/Spo enzyme from Streptococcus equisimilis. J. Bacteriol. 184, 2878–2888 (2002).
Van Nerom, K., Tamman, H., Takada, H., Hauryliuk, V. & Garcia-Pino, A. The Rel stringent factor from Thermus thermophilus: crystallization and X-ray analysis. Acta Crystallogr. F 75, 561–569 (2019).
Singal, B. et al. Crystallographic and solution structure of the N-terminal domain of the Rel protein from Mycobacterium tuberculosis. FEBS Lett. 591, 2323–2337 (2017).
Brown, A., Fernandez, I. S., Gordiyenko, Y. & Ramakrishnan, V. Ribosome-dependent activation of stringent control. Nature 534, 277–280 (2016).
Arenz, S. et al. The stringent factor RelA adopts an open conformation on the ribosome to stimulate ppGpp synthesis. Nucleic Acids Res. 44, 6471–6481 (2016).
Loveland, A. B. et al. Ribosome*RelA structures reveal the mechanism of stringent response activation. eLife 5, e17029 (2016).
Sun, D. et al. A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation responses. Nat. Struct. Mol. Biol. 17, 1188–1194 (2010).
Aravind, L. & Koonin, E. V. The HD domain defines a new superfamily of metal-dependent phosphohydrolases. Trends Biochem. Sci. 23, 469–472 (1998).
Manav, M. C. et al. Structural basis for (p)ppGpp synthesis by the Staphylococcus aureus small alarmone synthetase RelP. J. Biol. Chem. 293, 3254–3264 (2018).
Steinchen, W. et al. Structural and mechanistic divergence of the small (p)ppGpp synthetases RelP and RelQ. Sci. Rep. 8, 2195 (2018).
Takada, H. et al. Ribosome association primes the stringent factor Rel for recruitment of deacylated tRNA to ribosomal A-site. Preprint at bioRxiv https://doi.org/10.1101/2020.01.17.910273 (2020).
Takada, H. et al. The C-terminal RRM/ACT domain is crucial for fine-tuning the activation of ‘Long’ RelA-SpoT homolog enzymes by ribosomal complexes. Front. Microbiol. 11, 277–307 (2020).
Steinchen, W. et al. Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone. Proc. Natl Acad. Sci. USA 112, 13348–13353 (2015).
Okar, D. A. et al. PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate. Trends Biochem. Sci. 26, 30–35 (2001).
Gratani, F. L. et al. Regulation of the opposing (p)ppGpp synthetase and hydrolase activities in a bifunctional RelA/SpoT homologue from Staphylococcus aureus. PLoS Genet. 14, e1007514 (2018).
Ronneau, S. et al. Regulation of (p)ppGpp hydrolysis by a conserved archetypal regulatory domain. Nucleic Acids Res. 47, 843–854 (2019).
Kabsch, W. XDS. Acta Crystallogr D 66, 125–132 (2010).
Collaborative Computational Project, N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Terwilliger, T. C. et al. phenix.mr_rosetta: molecular replacement and model rebuilding with Phenix and Rosetta. J. Struct. Funct. Genomics 13, 81–90 (2012).
Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).
Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D 68, 368–380 (2012).
Talavera, A. et al. Phosphorylation decelerates conformational dynamics in bacterial translation elongation factors. Sci. Adv. 4, eaap9714 (2018).
Otosu, T., Ishii, K. & Tahara, T. Note: simple calibration of the counting-rate dependence of the timing shift of single photon avalanche diodes by photon interval analysis. Rev. Sci. Instrum. 84, 036105 (2013).
Schrimpf, W., Barth, A., Hendrix, J. & Lamb, D. C. PAM: a framework for integrated analysis of imaging, single-molecule, and ensemble fluorescence data. Biophys. J. 114, 1518–1528 (2018).
Hellenkamp, B. et al. Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study. Nat. Methods 15, 669 (2018).
Kudryavtsev, V. et al. Combining MFD and PIE for accurate single-pair Forster resonance energy transfer measurements. Chem. Phys. Chem. 13, 1060–1078 (2012).
Tomov, T. E. et al. Disentangling subpopulations in single-molecule FRET and ALEX experiments with photon distribution analysis. Biophys. J. 102, 1163–1173 (2012).
Antonik, M., Felekyan, S., Gaiduk, A. & Seidel, C. A. Separating structural heterogeneities from stochastic variations in fluorescence resonance energy transfer distributions via photon distribution analysis. J. Phys. Chem. B 110, 6970–6978 (2006).
Kalinin, S. et al. A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nat. Methods 9, 1218–1225 (2012).
Polikanov, Y. S., Steitz, T. A. & Innis, C. A. A proton wire to couple aminoacyl-tRNA accommodation and peptide-bond formation on the ribosome. Nat. Struct. Mol. Biol. 21, 787–793 (2014).
Antoun, A., Pavlov, M. Y., Tenson, T. & Ehrenberg, M. M. Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering. Biol. Proced. Online 6, 35–54 (2004).
Murina, V., Kasari, M., Hauryliuk, V. & Atkinson, G. C. Antibiotic resistance ABCF proteins reset the peptidyl transferase centre of the ribosome to counter translational arrest. Nucleic Acids Res. 46, 3753–3763 (2018).
Kudrin, P. et al. The ribosomal A-site finger is crucial for binding and activation of the stringent factor RelA. Nucleic Acids Res. 46, 1973–1983 (2018).
We acknowledge the use of the synchrotron-radiation facility at the SOLEIL synchrotron Gif-sur-Yvette, France, under proposals 20150717, 20160750 and 20170756. We also thank the staff from Swing, PROXIMA-1 and PROXIMA-2A beamlines at SOLEIL for assistance with data collection. This work was supported by grants from the Fonds National de Recherche Scientifique, nos. FNRS-EQP U.N043.17F, FRFS-WELBIO CR-2017S-03 and FNRS-PDR T.0066.18, and the Joint Programming Initiative on Antimicrobial Resistance (grant no. JPI-EC-AMR-R.8004.18-) to A.G.-P. The Program ‘Actions de Recherche Concertée’ 2016-2021 and Fonds d’Encouragement à la Recherche from the ULB, Fonds Jean Brachet and the Fondation Van Buren to A.G.-P.; the Molecular Infection Medicine Sweden, Swedish Research council (grant no. 2017-03783), and Ragnar Söderberg foundation fellowship to V.H.; J. Hendrix and J. Hofkens are grateful to the Research Foundation Flanders (FWO Vlaanderen, grant no. G0B4915N) and large infrastructure grant (no. ZW15_09 GOH6316N) and the KU Leuven Research Fund (no. C14/16/053); J.Hofkens thanks financial support of the Flemish government through long-term structural funding Methusalem (CASAS2, Meth/15/04). K.V.N. was supported by a PhD grant from the Fonds National de Recherche Scientifique FNRS-FRIA. N.V. acknowledges the Agency for Innovation by Science and Technology in Flanders for a PhD grant. H. Tamman was supported by a Chargé de Recherches fellowship from the FNRS (no. CR/DM-392). H. Takada was supported by the postdoctoral grant from the Umeå Centre for Microbial Research (UCMR).
The authors declare no competing interests.
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Tamman, H., Van Nerom, K., Takada, H. et al. A nucleotide-switch mechanism mediates opposing catalytic activities of Rel enzymes. Nat Chem Biol (2020). https://doi.org/10.1038/s41589-020-0520-2