A new family of bacterial ribosome hibernation factors

Abstract

To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage1,2. In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery3–6. Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon–EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation.

Rippling life on a dormant planet: hibernation of ribosomes, RNA polymerases, and other essential enzymes

“…The second lesson learned is that, despite the essential role of ribosome hibernation in cell survival, these proteins are not universally conserved across the evolutionary tree. Instead, bacteria and eukaryotes possess distinct sets of ribosome hibernation factors—including Stm1/Serbp1 ( Ben-Shem et al, 2011 ), Lso2/CCDC124 ( Wang et al, 2018 ), IFRD1/IFRD2 ( Brown et al, 2018 ), MDF1 ( Barandun et al, 2019 ), MDF2 ( Barandun et al, 2019 ), and Dap1b ( Leesch et al, 2023 ) in eukaryotes, and HPF/RaiA ( Agafonov et al, 1999 , 2001 ; Maki et al, 2000 ), RMF ( Wada et al, 1990 ), and Balon ( Helena-Bueno et al, 2024 ) in bacteria.…”

“…Similarly, ribosomes isolated from oocytes of frogs and zebrafish associate not only with the hibernation factors Serbp1(Hapb4) and Dapl1 in xenopus (frog) and dap1b in zebrafish, but also with eEF2 and the translation factor eIF5a ( Leesch et al, 2023 ). In bacteria, the elongation factor EF-Tu has been shown to bind hibernating ribosomes in metabolically inactive B. subtilis cells during spore formation ( Pereira et al, 2015 ), and in the γ-proteobacterium Psychrobacter urativorans where stress conditions trigger EF-Tu binding in addition to the hibernation factors HPF/RaiA and Balon ( Helena-Bueno et al, 2024 ). The common thread connecting all these non-hibernation factors that bind dormant ribosomes is the fact that they are all accessory factors in normal protein synthesis, making their binding to hibernating ribosomes highly unexpected ( Figure 3 ).…”

“…It remains unclear how conserved each family of ribosome hibernation factors is across species: whether they are broadly conserved across bacteria or eukaryotes, or if certain lineages of One difficulty in addressing this question lies in the much higher rate of sequence evolution for these proteins compared to other ribosomebinding proteins in a cell. This heightened rate of evolution makes it challenging to employ traditional approaches of homology search, such as simple Blast or even Markov Models-based search of homology, for the identification of their protein homologs across species (Helena-Bueno et al, 2024). However, the characteristic domain specificity of hibernation factors suggests that they emerged shortly after the split of bacteria and archaea-eukaryotes.…”

Ribosomal dormancy at the nexus of ribosome homeostasis and protein synthesis

“…[20] Recently, a new hibernation factor called Balon has been found in the bacteria Psychrobacter urativorans which occupies the ribosomal A-site along with translation elongation factor EFTu in dormant ribosomes formed under cold stress and stationary phase. [21] EFTu is involved in bringing the aminoacyl-tRNAs to the A-site during translation elongation. The study claims that Balon-EFTu-mediated dormant ribosome formation is a more wide-spread mechanism, as approximately 20% of bacterial species including Mycobacteria contain homologs of Balon.…”

“…The study claims that Balon-EFTu-mediated dormant ribosome formation is a more wide-spread mechanism, as approximately 20% of bacterial species including Mycobacteria contain homologs of Balon. [21] Future studies are required to understand the diversity of dormancy factors in prokaryotes.…”