Structure of a hibernating 100S ribosome reveals an inactive conformation of the ribosomal protein S1

Beckert, Bertrand; Turk, Martin; Czech, Andreas; Berninghausen, Otto; Beckmann, Roland; Ignatova, Zoya; Plitzko, Jürgen M.; Wilson, Daniel N.

doi:10.1038/s41564-018-0237-0

Cited by 107 publications

(117 citation statements)

References 49 publications

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“…Only the N-ter domain (NTD), bound to the ribosome, was recently resolved in complex with the ribosome (PDB IDs: 6H4N, 6BU8) [49,50]), and most of the other sequence regions of S1 are structurally uncharacterized (central domain and C-ter domain (CTD)). Biophysical studies suggest that the structure of S1 could be very elongated (up to 230 Å) [51], proposing a model of a bound NTD and a flexible CTD, which probes mRNA present in the cytosol.…”

Section: Localization and C-terminal Flexibility Of L31mentioning

confidence: 99%

“…7A) [9]. In ribosome hibernation, the dimeric interface is formed by the 30S subunit, while the 30S protein S2 of both ribosomes forms the core of the interface and 30S protein S1 is present in an inactive conformation [49]. The structure of the hibernated 100S ribosome has been solved by cryo-EM (PDB ID: 6H58) [49], but was not considered for mapping the cross-links due to its moderate resolution of 7.9 Å.…”

Section: Multimeric States Of Ribosomesmentioning

confidence: 99%

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Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

Tüting

Iacobucci

Ihling

et al. 2020

Preprint

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The ribosome is not only a highly complex molecular machine that executes translation according to the central dogma of molecular biology, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker diacetyl dibutyric urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-linkpairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolution E. coli ribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all crosslinks, resulting in an optimized E. coli ribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935.

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Section: Localization and C-terminal Flexibility Of L31mentioning

confidence: 99%

Section: Multimeric States Of Ribosomesmentioning

confidence: 99%

Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

Tüting

Iacobucci

Ihling

et al. 2020

Preprint

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“…In bacteria, several small ribosomal binding factors (RBFs) have been identified which inhibit translation and facilitate reversible formation of inactive 100S ribosome dimers Ortiz et al, 2010). These 100S dimers allow ribosomes to enter a hibernation state during stationary growth or stress phases (Beckert et al, 2017;Beckert et al, 2018;Matzov et al, 2017). Within hibernating bacterial ribosomes, RBFs (RMF, HPF, RaiA) bind the decoding center, occupy the mRNA binding channel, and block A and P tRNA sites (Ueta et al, 2008;Ueta et al, 2005;Yoshida et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Structure and function of yeast Lso2 and human CCDC124 bound to hibernating ribosomes

Wells

Buschauer

Mackens-Kiani

et al. 2020

Preprint

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150)Cells adjust to nutrient deprivation by reversible translational shut down. This is accompanied by maintaining inactive ribosomes in a hibernation state, where they are bound by proteins with inhibitory and protective functions. In eukaryotes, such a function was attributed to Stm1 (SERBP1 in mammals), and recently Lso2 (CCDC124 in mammals) was found to be involved in translational recovery after starvation from stationary phase. Here, we present cryo-electron microscopy (cryo-EM) structures of translationally inactive yeast and human ribosomes. We found Lso2/CCDC124 accumulating on idle ribosomes in the non-unrotated state, in contrast to Stm1/SERBP1-bound ribosomes, which display a rotated state. Lso2/CCDC124 bridges the decoding sites of the small with the GTPase-activating center of the large subunit. This position allows accommodation of the Dom34-dependent ribosome recycling system, which splits Lso2-containing but not Stm1-containing ribosomes. We propose a model in which Lso2 facilitates rapid translation reactivation by stabilizing the recycling-competent state of inactive ribosomes.

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“…The molecular model for the ribosomal proteins and rRNA core was based on the molecular model from the recent cryo-EM reconstructions of the E. coli 70S ribosome (PDB ID 6H4N 63 and 5MGP 38 . The models were rigid body fitted into the cryo-EM density map using UCSF Chimera followed by refinement in Coot 64 .…”

Section: Methodsmentioning

confidence: 99%

ResQ, a release factor-dependent ribosome rescue factor in the Gram-positive bacterium Bacillus subtilis

Shimokawa-Chiba

Müller

Fujiwara

et al. 2019

Preprint

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17Rescue of the ribosomes from dead-end translation complexes, such as those on 18 truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use 19 trans-translation for ribosome rescue, some Gram-negative species possess alternative 20 and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop 21 codon-independent polypeptide release. We now discover that the Gram-positive 22Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named ResQ. 23Genetic analysis shows that B. subtilis requires the function of either trans-translation or 24ResQ for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-EM 25 characterization demonstrates that ResQ binds to non-stop stalled ribosomes, recruits 26 homologous RF2, but not RF1, and induces its transition into an open active 27 conformation. Although ResQ is distinct from E. coli ArfA, they use convergent 28 strategies in terms of mode of action and expression regulation, indicating that many 29 bacteria may have evolved as yet unidentified ribosome rescue systems. 30 31 round of translation initiation, a failure in termination lowers the cellular capacity of 48 protein synthesis, unless dealt with by the cellular quality control mechanisms. Indeed, a 49 loss of function in the quality control machinery leads to an accumulation of dead-end 50 translation products, which was estimated to represent ~2-4% of the translation products 51 in E. coli 3 , and results in lethality 4,5 . 52Living organisms have evolved mechanisms that resolve non-productive 53 translation complexes produced by ribosome stalling on non-stop mRNAs. Such quality 54 control is also called ribosome rescue. In eukaryotic cells, the Dom34/Hbs1 complex, 55 together with Rli1/ABCE1, mediates ribosome rescue on truncated mRNAs 4,6,7 . In 56 bacteria, two distinct mechanisms operate in the resolution of non-stop nascent 57 chain-ribosome complexes, trans-translation and stop codon-independent peptide 58 release from the ribosome 4,5,8,9 . The latter mechanism can further be classified into two 59 classes, RF-dependent and RF-independent. The crucial player in trans-translation is the 60 transfer-messenger RNA (tmRNA), which is encoded by ssrA. tmRNA cooperates with 61 SmpB, which mediates ribosomal accommodation of tmRNA at the ribosomal A-site 10,11 . 62The tmRNA is composed of tRNA-and mRNA-like domains. The former can be 63 5 charged with alanine, which then accepts the non-stop peptide and is elongated further 64 according to the mRNA-like coding function of tmRNA until the built-in stop codon is 65 reached. The result is the formation of the non-stop polypeptide bearing an extra 66 ssrA-encoded sequence (15 amino acids in B. subtilis) and dissociation of the ribosome 67 from the non-stop mRNA. The SsrA tag sequence promotes proteolytic elimination of 68 the non-stop polypeptide via targeting to cellular proteases. Trans-translation is 69 essential for the growth of some bacteria 5,12 . The essentiality lies in the liberation o...

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Structure of a hibernating 100S ribosome reveals an inactive conformation of the ribosomal protein S1

Cited by 107 publications

References 49 publications

Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

Structure and function of yeast Lso2 and human CCDC124 bound to hibernating ribosomes

ResQ, a release factor-dependent ribosome rescue factor in the Gram-positive bacterium Bacillus subtilis

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