Biology subjects

Berninghausen, O.

Publications and source records attributed to Berninghausen, O..

At least 19 recordsLinked to original sources

bioRxiv2026

Dynamic architecture of the Rixosome reveals mechanism of activation and ITS2 processing

Eukaryotic ribosome assembly requires the coordinated processing and extensive remodeling of pre-rRNAs. During late nuclear maturation of the 60S subunit, sequential removal of the internal transcribed spacer 2 (ITS2) is initiated by endonucleolytic cleavage at site C2 by the conserved Las1 nuclease. Las1 acts together with the kinase Grc3 and the Rix1 complex to form the Rixosome, which also functions in transcriptional regulation. However, the assembly of the Rixosome, its recruitment to pre-ribosomes, and its activation for ITS2 cleavage remain unclear. Here, we present cryo-EM structures of the human LAS1 complex, two structures of the isolated Rixosome and nine transition states of Rix1-bound pre-60S particles from Schizosaccharomyces pombe. These structures reveal a dynamic Rixosome architecture in which the heterotetrameric Las1 complex engages one or two copies of the Rix1 complex. Rix1 binding is highly flexible in the human Rixosome but rigid in the yeast complex. The isolated yeast Rixosome remains inactive, but binding to the pre-60S particle triggers a structural rearrangement that allows for substrate engagement and activation of the nuclease. Together, our results define the dynamic architecture of the Rixosome and provide a structural framework for ITS2 processing during nuclear maturation of the eukaryotic 60S ribosomal subunit.

biochemistry↗

bioRxiv2026

Exopolysaccharide export complex PelBC of Pseudomonas aeruginosa attenuates the dynamics of the surrounding outer membrane

The opportunistic pathogen Pseudomonas aeruginosa ensures its survival by forming mechanically and chemically resistant biofilms, with cationic exopolysaccharide Pel as an abundant constituent of the structural matrix. Despite its biomedical relevance, the mechanisms of Pel synthesis and secretion via the trans-envelope protein machinery are not understood. Here, we examine the structure of the outer membrane export complex PelBC embedded in synthetic nanodiscs and polymer-extracted particles. Both environments preserve the unique architecture of the complex, where the beta-barrel PelB is capped with the dodecameric ring of PelC lipoproteins. Cryogenic electron microscopy shows that the polymer-extracted PelB beta-barrel is tightly associated with phospholipids and lipid A molecules, and the membrane-facing PelC ring may stabilize lipids of the periplasmic leaflet in defined positions. All-atom molecular dynamics simulation of PelBC in the asymmetric outer membrane of P. aeruginosa corroborate the structural findings and visualize how the essential C-terminal helix of PelC forms multiple electrostatic contacts with the periplasmic leaflet of the outer membrane. Those interactions reduce the lateral mobility of the lipids, stabilize the position of the ring at the interface and may guide folding and assembly of the polysaccharide export machinery.

microbiology↗

bioRxiv2025

Substrate-induced assembly and functional mechanism of the bacterial membrane protein insertase SecYEG-YidC

The Sec translocon and the YidC/Oxa1-type insertases universally mediate biogenesis of -helical membrane proteins, but the molecular basis of their cooperation has remained disputed. Recent discoveries of multi-subunit insertases assembled at the back of the translocon in fungi and higher eukaryotes have raised the question about the architecture and mechanism of the putative bacterial ortholog SecYEG-YidC. Here, we combine cryogenic electron microscopy with cell-free protein synthesis to visualize biogenesis of SecYEG/YidC-dependent multipass membrane protein NuoK. We demonstrate that the nascent chain of NuoK does not enter the lateral gate of SecYEG, but crosses over the translocon towards its back side, whereto YidC is recruited in the nascent chain-dependent manner. The SecY-YidC interface promotes folding of the transmembrane helices prior their insertion, in agreement with thermodynamic principles of membrane protein folding. YidC forms extensive contacts with the nascent chain, suggesting its key role in the insertion event. Our data provide detailed insights on the insertase machinery, suggest the evolutionary conservation of the gate-independent insertion route, and offer an expanded view on membrane protein biogenesis.

biochemistry↗

bioRxiv2024

Assembly and dynamics of the outer membrane exopolysaccharide transporter PelBC of Pseudomonas aeruginosa

The infamous opportunistic pathogen Pseudomonas aeruginosa enhances its virulence and antibiotic resistance upon formation of durable biofilms. The biofilm stability is mediated by its matrix built of secreted exopolysaccharides, eDNA, and structural proteins. Exopolysaccharides of P. aeruginosa - Pel, Psl and alginate - have the highest biomedical relevance, but the mechanisms behind their synthesis and secretion are poorly understood. Here, we employ cryogenic electron microscopy to resolve the 2.5 [A] structure of the outer membrane complex PelBC for Pel exopolysaccharide, which is uniquely composed of the membrane-embedded {beta}-barrel PelB and the asymmetrical ring of 12 lipoproteins PelC at the periplasmic interface. The assembly captured in the lipid-based nanodisc is stabilized by electrostatic contacts of PelC with the periplasmic loops of PelB and multiple interactions with PelB N-terminal helical domains. Within the membrane, the resolved acyl chains of the PelC lipoproteins are alternated by the tryptophan residues immersed into the lipid leaflet, thus offering a stable anchoring architecture. The highly anionic interior of the PelB {beta}-barrel is sealed by three loops at the extracellular side, where the short Plug-S loop is aligned with the periplasmic helical scaffold, being the potential gating element for the Pel exopolysaccharide tunneling. Molecular dynamic simulations of PelB in native-like membrane environments suggest that Plug-S is sufficiently flexible to open a tunnel, and so serve as a gate. The gating model is further supported by single-channel conductivity measurements, which identify two conductance states of PelB. Via mutational analysis we confirm that Plug-S mediates opening of a narrow tunnel, as required for the controlled exopolysaccharide transport. Our structural and functional analysis of the pathogenicity-relevant complex offer a detailed and comprehensive view on this unique machinery and suggest the route taken by the exopolysaccharide at the final secretion step.

biophysics↗

bioRxiv2024

The RNA helicase HrpA rescues collided ribosomes in E. coli

Although many antibiotics inhibit bacterial ribosomes, loss of known factors that rescue stalled ribosomes does not lead to robust antibiotic sensitivity in E. coli, suggesting the existence of additional mechanisms. Here, we show that the RNA helicase HrpA rescues stalled ribosomes in E. coli. Acting selectively on ribosomes that have collided, HrpA uses ATP hydrolysis to split stalled ribosomes into subunits. Cryo-EM structures reveal how HrpA simultaneously binds to two collided ribosomes, explaining its selectivity, and how its helicase module engages downstream mRNA, such that by exerting a pulling force on the mRNA, it would destabilize the stalled ribosome. These studies show that ribosome splitting is a conserved mechanism that allows proteobacteria to tolerate ribosome-targeting antibiotics.

molecular biology↗

bioRxiv2024

H/ACA snoRNP guides ribosomal RNA subdomain folding in a satellite particle before joining the core 90S pre-ribosome

During synthesis, modification and maturation of the ribosomal RNA, correct subdomain folding without additional guidance poses a major challenge. An essential H/ACA snoRNP, snR30, was observed to be outsourced from the core of the 90S, the earliest known pre-ribosome, to form a "satellite particle". This particle interacts with the so-called 18S rRNA platform subdomain, guiding its independent folding through localized structural interactions. Once this subdomain achieves its proper conformation, it is integrated into the core pre-ribosome, contributing to the overall maturation of the 18S rRNA. By temporarily segregating the folding process within a specialized satellite particle, the correct assembly of individual components is facilitated, thereby reducing the risk of errors during the complex process of ribosome assembly. In a broader sense, molecular outsourcing in cellular processes, where transient subcomplexes handle specific tasks before rejoining the main assembly line, can enhance efficiency and accuracy of complex molecular processes like ribosome biogenesis.

biochemistry↗

bioRxiv2023

B. subtilis MutS2 splits stalled ribosomesinto subunits without mRNA cleavage

Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains and reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation by the ribosome quality control pathway. Notably, we see no evidence of mRNA cleavage by MutS2, nor does it promote ribosome rescue by tmRNA as SmrB cleavage does in E. coli. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in various bacteria.

molecular biology↗

bioRxiv2022

Concurrent remodelling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase

Biogenesis intermediates of nucleolar ribosomal 60S precursor particles undergo a number of structural maturation steps before they transit to the nucleoplasm and are finally exported into the cytoplasm. The AAA+-ATPase Rea1 participates in the nucleolar exit by releasing the Ytm1-Erb1 heterodimer from the evolving pre-60S particle. Here, we show that the DEAD-box RNA helicase Spb4 with its interacting partner Rrp17 is further integrated into this maturation event. Spb4 binds to a specific class of late nucleolar pre-60S intermediates, whose cryo-EM structure revealed how its helicase activity facilitates melting and restructuring of 25S rRNA helices H62 and H63/H63a prior to Ytm1-Erb1 release. In vitro maturation of such Spb4-enriched pre-60S particles, incubated with purified Rea1 and its associated pentameric Rix1-complex in the presence of ATP, combined with cryo-EM analysis depicted the details of the Rea1-dependent large-scale pre-ribosomal remodelling. Our structural insights unveil how the Rea1 ATPase and Spb4 helicase remodel late nucleolar pre-60S particles by rRNA restructuring and dismantling of a network of several ribosomal assembly factors.

biochemistry↗

bioRxiv2022

Clearing of ribosome collisions by the ribosome quality control trigger complex RQT

After translational stalls, colliding eukaryotic ribosomes are cleared through dissociation into subunits by the ribosome quality control trigger complex, RQT, by an unknown mechanism. Here we show that RQT requires accessible mRNA and the presence of a neighboring ribosome. Cryo-EM of several RQT-ribosome complexes revealed the structural basis of splitting: RQT engages the 40S subunit of the lead ribosome and can switch between two conformations. We propose a mechanistic model in which the Slh1 helicase subunit of RQT applies a pulling force on the mRNA, causing destabilizing conformational changes of the 40S subunit. The collided ribosome functions as a ram or giant wedge, ultimately resulting in subunit dissociation. Our findings provide a first conceptual framework for a helicase driven ribosomal splitting mechanism. One-Sentence SummaryRQT clears collided ribosomes by pulling mRNA to trigger destabilizing conformational transitions for subunit dissociation.

biochemistry↗

bioRxiv2021

Recognition and deubiquitination of free 40S for translational reset by Otu2

In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not41,2 and deubiquitinated by the deubiquitination enzyme Otu2 upon ribosomal subunit recycling3. Despite its importance for general efficiency of translation the exact role and structural basis for this specific translational reset are only poorly understood. Here we present biochemical and structural data showing that Otu2 can engage the recycled 40S subunit together with the recycling factors ABCE1 and Tma64 immediately after 60S dissociation for mRNA recycling, and that it dissociates before 48S initiation complex formation. A combined structural analysis of Otu2 and Otu2-40S complexes by X-ray crystallography, AlphaFold2 prediction4 and cryo-EM revealed how Otu2 can specifically be recruited to the 40S, but not to the 80S ribosome, for removal of the eS7-bound ubiquitin moiety. Here, interactions of the largely helical N-terminal domain of Otu2 to sites that are masked and therefore inaccessible in the 80S ribosome are of crucial importance. Collectively, we provide the structural basis for the Otu2 driven deubiquitination step providing a first mechanistic understanding of this translational reset step during ribosome recycling/(re)initiation.

molecular biology↗

bioRxiv2021

Ribosome collisions in bacteria promote ribosome rescue by triggering mRNA cleavage by SmrB

Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes. In a genetic screen in E. coli, we discovered a novel rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNAs) to rescue upstream ribosomes. SmrB is recruited by ribosome collisions; cryo-EM structures of collided disomes from E. coli and B. subtilis reveal a distinct and conserved arrangement of the individual ribosomes and the composite SmrB binding site. These findings reveal the underlying mechanism by which ribosome collisions trigger ribosome rescue in bacteria.

molecular biology↗

bioRxiv2021

A distinct assembly pathway of the human 39S late pre-mitoribosome

Assembly of the mitoribosome is largely enigmatic and involves numerous assembly factors. However, little is known about their function and the architectural transitions of the pre-ribosomal intermediates. Here, we solved cryo-EM structures of the human 39S large subunit pre-ribosomes, representing four distinct late state intermediates. Besides the MALSU1 complex, we identified several assembly factors, including the DDX28 helicase, MRM3, GTPBP10 and the NSUN4-mTERF4 complex, all of which keep the 16S rRNA in immature conformations. The late transitions mainly involved rRNA domains IV and V, which form the central protuberance, the intersubunit side and the peptidyltransferase center of the 39S subunit. Unexpectedly, we found deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly. Taken together, our study provides an architectural inventory of the distinct late assembly phase of the human 39S mitoribosome.

biochemistry↗

bioRxiv2020

Structure of Gcn1 bound to stalled and colliding 80S ribosomes

Protein synthesis is essential to cells and requires a constant supply of nutrients. Amino acid starvation leads to accumulation of uncharged tRNAs that promote ribosomal stalling, which is sensed by the protein kinase Gcn2, together with its effector proteins, Gcn1 and Gcn20. Activation of Gcn2 phosphorylates eIF2, leading to a global repression of translation. Fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, which is a negative regulator of Gcn2. Despite the wealth of biochemical data, structures of Gcn proteins on the ribosome have remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the A-site region of the lead ribosome. The lead ribosome is stalled in a non-rotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, as well as Rbg2/Gir2 located in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings provide a structural basis for Rbg2/Gir2 repression of Gcn2, and also reveal that colliding disomes are the substrate for Gcn1 binding, which has important implications not only for Gcn2-activated stress responses, but also for general ribosome quality control (RQC) pathways.

biochemistry↗

bioRxiv2020

Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2

SARS-CoV-2 is the causative agent of the current COVID-19 pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1) which suppresses host gene expression by ribosome association via an unknown mechanism. Here, we show that Nsp1 from SARS-CoV-2 binds to 40S and 80S ribosomes, resulting in shutdown of capped mRNA translation both in vitro and in cells. Structural analysis by cryo-electron microscopy (cryo-EM) of in vitro reconstituted Nsp1-40S and of native human Nsp1-ribosome complexes revealed that the Nsp1 C-terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks RIG-I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.

molecular biology↗

bioRxiv2020

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

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.

molecular biology↗

bioRxiv2019

The Ccr4-Not complex monitors the translating ribosome for codon optimality

Control of mRNA decay rate is intimately connected to translation elongation but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. Using a combination of cryo-electron microscopy, ribosome profiling and mRNA stability assays we show recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site. This interaction only occurs when the ribosome lacks accommodated A-site tRNA, indicative of low codon optimality. Loss of Not5 results in the inability of the mRNA degradation machinery to sense codon optimality. Our analysis elucidates a physical link between the Ccr4-Not complex and the ribosome providing mechanistic insight into the coupling of decoding efficiency with mRNA stability.

molecular biology↗

bioRxiv2019

Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

biochemistry↗

bioRxiv2019

Structural and mutational analysis of the ribosome-arresting human XBP1u

XBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 [A] cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the upper part of the ribosomal exit tunnel and causes a subtle distortion of the peptidyl transferase center, explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 10 out of 21 mutagenized positions in the XBP1u AP are optimal with respect to translational arrest activity. Thus, XBP1u has evolved to induce an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel and thereby serving its central function in the UPR.

biochemistry↗