Protein synthesis (translation) consumes a substantial proportion of cellular resources, prompting specialized mechanisms to reduce translation under adverse conditions. Ribosome inactivation often involves ribosome-interacting proteins. In both bacteria and eukaryotes, various ribosome-interacting proteins facilitate ribosome dimerization or hibernation, and/or prevent ribosomal subunits from associating, enabling the organisms to adapt to stress. Despite extensive studies on bacteria and eukaryotes, understanding factor-mediated ribosome dimerization or anti-association in archaea remains elusive. Here, we present cryo-electron microscopy structures of an archaeal 30S dimer complexed with an archaeal ribosome dimerization factor (designated aRDF), from Pyrococcus furiosus, resolved at a resolution of 3.2 Å. The complex features two 30S subunits stabilized by aRDF homodimers in a unique head-to-body architecture, which differs from the disome architecture observed during hibernation in bacteria and eukaryotes. aRDF interacts directly with eS32 ribosomal protein, which is essential for subunit association. The binding mode of aRDF elucidates its anti-association properties, which prevent the assembly of archaeal 70S ribosomes.

Central European Institute of Technology Masaryk University Kamenice 5 Brno 625 00 Czech Republic

Department of Biology Faculty of Science Niigata University 8050 Ikarashi 2 no cho Niigata 950 2181 Japan

Department of Bioscience and Biotechnology Faculty of Agriculture Kyushu University 744 Motooka Nishi ku Fukuoka 819 0395 Japan

Zobrazit více v PubMed

Green  R., Noller  H.F.  Ribosomes and translation. Annu. Rev. Biochem.  1997; 66:679–716. PubMed

Russell  J.B., Cook  G.M.  Energetics of bacterial growth: balance of anabolic and catabolic reactions. Microbiol. Rev.  1995; 59:48–62. PubMed PMC

Njenga  R., Boele  J., Ozturk  Y., Koch  H.G.  Coping with stress: how bacteria fine-tune protein synthesis and protein transport. J. Biol. Chem.  2023; 299:105163. PubMed PMC

Starosta  A.L., Lassak  J., Jung  K., Wilson  D.N.  The bacterial translation stress response. FEMS Microbiol. Rev.  2014; 38:1172–1201. PubMed PMC

Prossliner  T., Skovbo Winther  K., Sorensen  M.A., Gerdes  K.  Ribosome hibernation. Annu. Rev. Genet.  2018; 52:321–348. PubMed

Kumar  N., Sharma  S., Kaushal  P.S.  Cryo-EM structure of the mycobacterial 70S ribosome in complex with ribosome hibernation promotion factor RafH. Nat. Commun.  2024; 15:638. PubMed PMC

Polikanov  Y.S., Blaha  G.M., Steitz  T.A.  How hibernation factors RMF, HPF, and YfiA turn off protein synthesis. Science. 2012; 336:915–918. PubMed PMC

Helena-Bueno  K., Rybak  M.Y., Ekemezie  C.L., Sullivan  R., Brown  C.R., Dingwall  C., Basle  A., Schneider  C., Connolly  J.P.R., Blaza  J.N.  et al. .  A new family of bacterial ribosome hibernation factors. Nature. 2024; 626:1125–1132. PubMed PMC

Beckert  B., Turk  M., Czech  A., Berninghausen  O., Beckmann  R., Ignatova  Z., Plitzko  J.M., Wilson  D.N.  Structure of a hibernating 100S ribosome reveals an inactive conformation of the ribosomal protein S1. Nat. Microbiol.  2018; 3:1115–1121. PubMed

Flygaard  R.K., Boegholm  N., Yusupov  M., Jenner  L.B.  Cryo-EM structure of the hibernating Thermus thermophilus 100S ribosome reveals a protein-mediated dimerization mechanism. Nat. Commun.  2018; 9:4179. PubMed PMC

Beckert  B., Abdelshahid  M., Schafer  H., Steinchen  W., Arenz  S., Berninghausen  O., Beckmann  R., Bange  G., Turgay  K., Wilson  D.N.  Structure of the Bacillus subtilis hibernating 100S ribosome reveals the basis for 70S dimerization. EMBO J.  2017; 36:2061–2072. PubMed PMC

Franken  L.E., Oostergetel  G.T., Pijning  T., Puri  P., Arkhipova  V., Boekema  E.J., Poolman  B., Guskov  A.  A general mechanism of ribosome dimerization revealed by single-particle cryo-electron microscopy. Nat. Commun.  2017; 8:722. PubMed PMC

Khusainov  I., Vicens  Q., Ayupov  R., Usachev  K., Myasnikov  A., Simonetti  A., Validov  S., Kieffer  B., Yusupova  G., Yusupov  M.  et al. .  Structures and dynamics of hibernating ribosomes from Staphylococcus aureus mediated by intermolecular interactions of HPF. EMBO J.  2017; 36:2073–2087. PubMed PMC

Matzov  D., Aibara  S., Basu  A., Zimmerman  E., Bashan  A., Yap  M.F., Amunts  A., Yonath  A.E.  The cryo-EM structure of hibernating 100S ribosome dimer from pathogenic Staphylococcus aureus. Nat. Commun.  2017; 8:723. PubMed PMC

Ortiz  J.O., Brandt  F., Matias  V.R., Sennels  L., Rappsilber  J., Scheres  S.H., Eibauer  M., Hartl  F.U., Baumeister  W.  Structure of hibernating ribosomes studied by cryoelectron tomography in vitro and in situ. J. Cell Biol.  2010; 190:613–621. PubMed PMC

Kato  T., Yoshida  H., Miyata  T., Maki  Y., Wada  A., Namba  K.  Structure of the 100S ribosome in the hibernation stage revealed by electron cryomicroscopy. Structure. 2010; 18:719–724. PubMed

Wells  J.N., Buschauer  R., Mackens-Kiani  T., Best  K., Kratzat  H., Berninghausen  O., Becker  T., Gilbert  W., Cheng  J., Beckmann  R.  Structure and function of yeast Lso2 and human CCDC124 bound to hibernating ribosomes. PLoS Biol.  2020; 18:e3000780. PubMed PMC

Ben-Shem  A., Garreau de Loubresse  N., Melnikov  S., Jenner  L., Yusupova  G., Yusupov  M.  The structure of the eukaryotic ribosome at 3.0 A resolution. Science. 2011; 334:1524–1529. PubMed

McLaren  M., Conners  R., Isupov  M.N., Gil-Diez  P., Gambelli  L., Gold  V.A.M., Walter  A., Connell  S.R., Williams  B., Daum  B.  CryoEM reveals that ribosomes in microsporidian spores are locked in a dimeric hibernating state. Nat. Microbiol.  2023; 8:1834–1845. PubMed PMC

McCutcheon  J.P., Agrawal  R.K., Philips  S.M., Grassucci  R.A., Gerchman  S.E., Clemons  W.M., Ramakrishnan  V., Frank  J.  Location of translational initiation factor IF3 on the small ribosomal subunit. Proc. Natl. Acad. Sci. U.S.A.  1999; 96:4301–4306. PubMed PMC

Petrelli  D., LaTeana  A., Garofalo  C., Spurio  R., Pon  C.L., Gualerzi  C.O.  Translation initiation factor IF3: two domains, five functions, one mechanism. EMBO J.  2001; 20:4560–4569. PubMed PMC

Hussain  T., Llacer  J.L., Wimberly  B.T., Kieft  J.S., Ramakrishnan  V.  Large-scale movements of IF3 and tRNA during bacterial translation initiation. Cell. 2016; 167:133–144. PubMed PMC

Fabbretti  A., Pon  C.L., Hennelly  S.P., Hill  W.E., Lodmell  J.S., Gualerzi  C.O.  The real-time path of translation factor IF3 onto and off the ribosome. Mol. Cell. 2007; 25:285–296. PubMed

Milon  P., Konevega  A.L., Gualerzi  C.O., Rodnina  M.V.  Kinetic checkpoint at a late step in translation initiation. Mol. Cell. 2008; 30:712–720. PubMed

MacDougall  D.D., Gonzalez  R.L.  Jr  Translation initiation factor 3 regulates switching between different modes of ribosomal subunit joining. J. Mol. Biol.  2015; 427:1801–1818. PubMed PMC

Kolupaeva  V.G., Unbehaun  A., Lomakin  I.B., Hellen  C.U., Pestova  T.V.  Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association. RNA. 2005; 11:470–486. PubMed PMC

des Georges  A., Dhote  V., Kuhn  L., Hellen  C.U., Pestova  T.V., Frank  J., Hashem  Y.  Structure of mammalian eIF3 in the context of the 43S preinitiation complex. Nature. 2015; 525:491–495. PubMed PMC

Yaeshima  C., Murata  N., Ishino  S., Sagawa  I., Ito  K., Uchiumi  T.  A novel ribosome-dimerization protein found in the hyperthermophilic archaeon Pyrococcus furiosus using ribosome-associated proteomics. Biochem. Biophys. Res. Commun.  2022; 593:116–121. PubMed

Shiraishi  M., Ishino  S., Yamagami  T., Egashira  Y., Kiyonari  S., Ishino  Y.  A novel endonuclease that may be responsible for damaged DNA base repair in Pyrococcus furiosus. Nucleic Acids Res.  2015; 43:2853–2863. PubMed PMC

Iacobucci  C., Gotze  M., Ihling  C.H., Piotrowski  C., Arlt  C., Schafer  M., Hage  C., Schmidt  R., Sinz  A.  A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein–protein interactions. Nat. Protoc.  2018; 13:2864–2889. PubMed

Mastronarde  D.N.  Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol.  2005; 152:36–51. PubMed

Zheng  S.Q., Palovcak  E., Armache  J.P., Verba  K.A., Cheng  Y., Agard  D.A.  MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods. 2017; 14:331–332. PubMed PMC

Grant  T., Rohou  A., Grigorieff  N.  cisTEM, user-friendly software for single-particle image processing. eLife. 2018; 7:e35383. PubMed PMC

Rohou  A., Grigorieff  N.  CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol.  2015; 192:216–221. PubMed PMC

Grigorieff  N.  Frealign: an exploratory tool for single-particle cryo-EM. Methods Enzymol.  2016; 579:191–226. PubMed PMC

Armache  J.P., Anger  A.M., Marquez  V., Franckenberg  S., Frohlich  T., Villa  E., Berninghausen  O., Thomm  M., Arnold  G.J., Beckmann  R.  et al. .  Promiscuous behaviour of archaeal ribosomal proteins: implications for eukaryotic ribosome evolution. Nucleic Acids Res.  2013; 41:1284–1293. PubMed PMC

Pettersen  E.F., Goddard  T.D., Huang  C.C., Couch  G.S., Greenblatt  D.M., Meng  E.C., Ferrin  T.E.  UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem.  2004; 25:1605–1612. PubMed

Heymann  J.B., Belnap  D.M.  Bsoft: image processing and molecular modeling for electron microscopy. J. Struct. Biol.  2007; 157:3–18. PubMed

Kazan  R., Bourgeois  G., Lazennec-Schurdevin  C., Larquet  E., Mechulam  Y., Coureux  P.D., Schmitt  E.  Role of aIF5B in archaeal translation initiation. Nucleic Acids Res.  2022; 50:6532–6548. PubMed PMC

Jumper  J., Evans  R., Pritzel  A., Green  T., Figurnov  M., Ronneberger  O., Tunyasuvunakool  K., Bates  R., Zidek  A., Potapenko  A.  et al. .  Highly accurate protein structure prediction with AlphaFold. Nature. 2021; 596:583–589. PubMed PMC

Emsley  P., Cowtan  K.  Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr.  2004; 60:2126–2132. PubMed

Liebschner  D., Afonine  P.V., Baker  M.L., Bunkoczi  G., Chen  V.B., Croll  T.I., Hintze  B., Hung  L.W., Jain  S., McCoy  A.J.  et al. .  Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol.  2019; 75:861–877. PubMed PMC

Williams  C.J., Headd  J.J., Moriarty  N.W., Prisant  M.G., Videau  L.L., Deis  L.N., Verma  V., Keedy  D.A., Hintze  B.J., Chen  V.B.  et al. .  MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci.  2018; 27:293–315. PubMed PMC

Meng  E.C., Goddard  T.D., Pettersen  E.F., Couch  G.S., Pearson  Z.J., Morris  J.H., Ferrin  T.E.  UCSF ChimeraX: tools for structure building and analysis. Protein Sci.  2023; 32:e4792. PubMed PMC

Krissinel  E., Henrick  K.  Inference of macromolecular assemblies from crystalline state. J. Mol. Biol.  2007; 372:774–797. PubMed

Agirre  J., Atanasova  M., Bagdonas  H., Ballard  C.B., Basle  A., Beilsten-Edmands  J., Borges  R.J., Brown  D.G., Burgos-Marmol  J.J., Berrisford  J.M.  et al. .  The CCP4 suite: integrative software for macromolecular crystallography. Acta Crystallogr. D Struct. Biol.  2023; 79:449–461. PubMed PMC

de Vienne  D.M.  Lifemap: exploring the entire tree of life. PLoS Biol.  2016; 14:e2001624. PubMed PMC

Letunic  I., Bork  P.  Interactive Tree Of Life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res.  2016; 44:W242–W245. PubMed PMC

Johnson  M., Zaretskaya  I., Raytselis  Y., Merezhuk  Y., McGinnis  S., Madden  T.L.  NCBI BLAST: a better web interface. Nucleic Acids Res.  2008; 36:W5–W9. PubMed PMC

The UniProt Consortium  UniProt: the Universal Protein knowledgebase in 2023. Nucleic Acids Res.  2023; 51:D523–D531. PubMed PMC

van Kempen  M., Kim  S.S., Tumescheit  C., Mirdita  M., Lee  J., Gilchrist  C.L.M., Soding  J., Steinegger  M.  Fast and accurate protein structure search with Foldseek. Nat. Biotechnol.  2024; 42:243–246. PubMed PMC

Zhang  C., Zheng  W., Mortuza  S.M., Li  Y., Zhang  Y.  DeepMSA: constructing deep multiple sequence alignment to improve contact prediction and fold-recognition for distant-homology proteins. Bioinformatics. 2020; 36:2105–2112. PubMed PMC

Crooks  G.E., Hon  G., Chandonia  J.M., Brenner  S.E.  WebLogo: a sequence logo generator. Genome Res.  2004; 14:1188–1190. PubMed PMC

Ban  N., Beckmann  R., Cate  J.H., Dinman  J.D., Dragon  F., Ellis  S.R., Lafontaine  D.L., Lindahl  L., Liljas  A., Lipton  J.M.  et al. .  A new system for naming ribosomal proteins. Curr. Opin. Struct. Biol.  2014; 24:165–169. PubMed PMC

Yusupov  M.M., Yusupova  G.Z., Baucom  A., Lieberman  K., Earnest  T.N., Cate  J.H., Noller  H.F.  Crystal structure of the ribosome at 5.5 Å resolution. Science. 2001; 292:883–896. PubMed

Khusainov  I., Fatkhullin  B., Pellegrino  S., Bikmullin  A., Liu  W.T., Gabdulkhakov  A., Shebel  A.A., Golubev  A., Zeyer  D., Trachtmann  N.  et al. .  Mechanism of ribosome shutdown by RsfS in Staphylococcus aureus revealed by integrative structural biology approach. Nat. Commun.  2020; 11:1656. PubMed PMC

Gartmann  M., Blau  M., Armache  J.P., Mielke  T., Topf  M., Beckmann  R.  Mechanism of eIF6-mediated inhibition of ribosomal subunit joining. J. Biol. Chem.  2010; 285:14848–14851. PubMed PMC

Weis  F., Giudice  E., Churcher  M., Jin  L., Hilcenko  C., Wong  C.C., Traynor  D., Kay  R.R., Warren  A.J.  Mechanism of eIF6 release from the nascent 60S ribosomal subunit. Nat. Struct. Mol. Biol.  2015; 22:914–919. PubMed PMC

Madoori  P.K., Agustiandari  H., Driessen  A.J., Thunnissen  A.M.  Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognition. EMBO J.  2009; 28:156–166. PubMed PMC

Belitsky  B.R., Sonenshein  A.L.  Genome-wide identification of Bacillus subtilis CodY-binding sites at single-nucleotide resolution. Proc. Natl. Acad. Sci. U.S.A.  2013; 110:7026–7031. PubMed PMC

Coureux  P.D., Lazennec-Schurdevin  C., Bourcier  S., Mechulam  Y., Schmitt  E.  Cryo-EM study of an archaeal 30S initiation complex gives insights into evolution of translation initiation. Commun. Biol.  2020; 3:58. PubMed PMC

Hentschel  J., Burnside  C., Mignot  I., Leibundgut  M., Boehringer  D., Ban  N.  The complete structure of the Mycobacteriumsmegmatis 70S ribosome. Cell Rep.  2017; 20:149–160. PubMed

Trosch  R., Willmund  F.  The conserved theme of ribosome hibernation: from bacteria to chloroplasts of plants. Biol. Chem.  2019; 400:879–893. PubMed

Juszkiewicz  S., Chandrasekaran  V., Lin  Z., Kraatz  S., Ramakrishnan  V., Hegde  R.S.  ZNF598 is a quality control sensor of collided ribosomes. Mol. Cell. 2018; 72:469–481. PubMed PMC

Narita  M., Denk  T., Matsuo  Y., Sugiyama  T., Kikuguchi  C., Ito  S., Sato  N., Suzuki  T., Hashimoto  S., Machova  I.  et al. .  A distinct mammalian disome collision interface harbors K63-linked polyubiquitination of uS10 to trigger hRQT-mediated subunit dissociation. Nat. Commun.  2022; 13:6411. PubMed PMC

Flugel  T., Schacherl  M., Unbehaun  A., Schroeer  B., Dabrowski  M., Burger  J., Mielke  T., Sprink  T., Diebolder  C.A., Guillen Schlippe  Y.V.  et al. .  Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM. Nat. Commun.  2024; 15:1756. PubMed PMC

Barandun  J., Hunziker  M., Vossbrinck  C.R., Klinge  S.  Evolutionary compaction and adaptation visualized by the structure of the dormant microsporidian ribosome. Nat. Microbiol.  2019; 4:1798–1804. PubMed PMC

Yi  S.H., Petrychenko  V., Schliep  J.E., Goyal  A., Linden  A., Chari  A., Urlaub  H., Stark  H., Rodnina  M.V., Adio  S.  et al. .  Conformational rearrangements upon start codon recognition in human 48S translation initiation complex. Nucleic Acids Res.  2022; 50:5282–5298. PubMed PMC

Zavialov  A.V., Hauryliuk  V.V., Ehrenberg  M.  Splitting of the posttermination ribosome into subunits by the concerted action of RRF and EF-G. Mol. Cell. 2005; 18:675–686. PubMed

Becker  T., Franckenberg  S., Wickles  S., Shoemaker  C.J., Anger  A.M., Armache  J.P., Sieber  H., Ungewickell  C., Berninghausen  O., Daberkow  I.  et al. .  Structural basis of highly conserved ribosome recycling in eukaryotes and archaea. Nature. 2012; 482:501–506. PubMed PMC

Rappaport  H.B., Oliverio  A.M.  Lessons from extremophiles: functional adaptations and genomic innovations across the eukaryotic tree of life. Genome Biol. Evol.  2024; 16:evae160. PubMed PMC

Kobayashi  K., Saito  K., Ishitani  R., Ito  K., Nureki  O.  Structural basis for translation termination by archaeal RF1 and GTP-bound EF1alpha complex. Nucleic Acids Res.  2012; 40:9319–9328. PubMed PMC

Kobayashi  K., Kikuno  I., Kuroha  K., Saito  K., Ito  K., Ishitani  R., Inada  T., Nureki  O.  Structural basis for mRNA surveillance by archaeal Pelota and GTP-bound EF1alpha complex. Proc. Natl. Acad. Sci. U.S.A.  2010; 107:17575–17579. PubMed PMC

Grunberger  F., Schmid  G., El Ahmad  Z., Fenk  M., Vogl  K., Reichelt  R., Hausner  W., Urlaub  H., Lenz  C., Grohmann  D.  Uncovering the temporal dynamics and regulatory networks of thermal stress response in a hyperthermophile using transcriptomics and proteomics. mBio. 2023; 14:e0217423. PubMed PMC