Publications by authors named "Otto Berninghausen"

75 Publications

Structure of Gcn1 bound to stalled and colliding 80S ribosomes.

Proc Natl Acad Sci U S A 2021 Apr;118(14)

Institute for Biochemistry and Molecular Biology, University of Hamburg, 20146 Hamburg, Germany;

The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has 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 P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 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 reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2022756118DOI Listing
April 2021

Structure of the Maturing 90S Pre-ribosome in Association with the RNA Exosome.

Mol Cell 2021 01 15;81(2):293-303.e4. Epub 2020 Dec 15.

Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. Electronic address:

Ribosome assembly is catalyzed by numerous trans-acting factors and coupled with irreversible pre-rRNA processing, driving the pathway toward mature ribosomal subunits. One decisive step early in this progression is removal of the 5' external transcribed spacer (5'-ETS), an RNA extension at the 18S rRNA that is integrated into the huge 90S pre-ribosome structure. Upon endo-nucleolytic cleavage at an internal site, A, the 5'-ETS is separated from the 18S rRNA and degraded. Here we present biochemical and cryo-electron microscopy analyses that depict the RNA exosome, a major 3'-5' exoribonuclease complex, in a super-complex with the 90S pre-ribosome. The exosome is docked to the 90S through its co-factor Mtr4 helicase, a processive RNA duplex-dismantling helicase, which strategically positions the exosome at the base of 5'-ETS helices H9-H9', which are dislodged in our 90S-exosome structures. These findings suggest a direct role of the exosome in structural remodeling of the 90S pre-ribosome to drive eukaryotic ribosome synthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.11.009DOI Listing
January 2021

Architecture of the active post-translational Sec translocon.

EMBO J 2021 Feb 11;40(3):e105643. Epub 2020 Dec 11.

Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany.

In eukaryotes, most secretory and membrane proteins are targeted by an N-terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein-conducting channel (PCC). In the post-translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre-opened state. Here, we present a cryo-EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N-terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C-terminus of Sec63. Together, we obtained a near-complete and integrated model of the active Sec complex.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.2020105643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849165PMC
February 2021

A structural inventory of native ribosomal ABCE1-43S pre-initiation complexes.

EMBO J 2021 01 8;40(1):e105179. Epub 2020 Dec 8.

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

In eukaryotic translation, termination and ribosome recycling phases are linked to subsequent initiation of a new round of translation by persistence of several factors at ribosomal sub-complexes. These comprise/include the large eIF3 complex, eIF3j (Hcr1 in yeast) and the ATP-binding cassette protein ABCE1 (Rli1 in yeast). The ATPase is mainly active as a recycling factor, but it can remain bound to the dissociated 40S subunit until formation of the next 43S pre-initiation complexes. However, its functional role and native architectural context remains largely enigmatic. Here, we present an architectural inventory of native yeast and human ABCE1-containing pre-initiation complexes by cryo-EM. We found that ABCE1 was mostly associated with early 43S, but also with later 48S phases of initiation. It adopted a novel hybrid conformation of its nucleotide-binding domains, while interacting with the N-terminus of eIF3j. Further, eIF3j occupied the mRNA entry channel via its ultimate C-terminus providing a structural explanation for its antagonistic role with respect to mRNA binding. Overall, the native human samples provide a near-complete molecular picture of the architecture and sophisticated interaction network of the 43S-bound eIF3 complex and the eIF2 ternary complex containing the initiator tRNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.2020105179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780240PMC
January 2021

Structural basis for the final steps of human 40S ribosome maturation.

Nature 2020 11 18;587(7835):683-687. Epub 2020 Nov 18.

Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Eukaryotic ribosomes consist of a small 40S and a large 60S subunit that are assembled in a highly coordinated manner. More than 200 factors ensure correct modification, processing and folding of ribosomal RNA and the timely incorporation of ribosomal proteins. Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3 by the endonuclease NOB1. Previous structures of human 40S precursors have shown that NOB1 is kept in an inactive state by its partner PNO1. The final maturation events, including the activation of NOB1 for the decisive rRNA-cleavage step and the mechanisms driving the dissociation of the last biogenesis factors have, however, remained unresolved. Here we report five cryo-electron microscopy structures of human 40S subunit precursors, which describe the compositional and conformational progression during the final steps of 40S assembly. Our structures explain the central role of RIOK1 in the displacement and dissociation of PNO1, which in turn allows conformational changes and activation of the endonuclease NOB1. In addition, we observe two factors, eukaryotic translation initiation factor 1A domain-containing protein (EIF1AD) and leucine-rich repeat-containing protein 47 (LRRC47), which bind to late pre-40S particles near RIOK1 and the central rRNA helix 44. Finally, functional data shows that EIF1AD is required for efficient assembly factor recycling and 18S-E processing. Our results thus enable a detailed understanding of the last steps in 40S formation in human cells and, in addition, provide evidence for principal differences in small ribosomal subunit formation between humans and the model organism Saccharomyces cerevisiae.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2929-xDOI Listing
November 2020

90 pre-ribosome transformation into the primordial 40 subunit.

Science 2020 09;369(6510):1470-1476

Gene Center, Department of Biochemistry, University of Munich, 81377 Munich, Germany.

Production of small ribosomal subunits initially requires the formation of a 90 precursor followed by an enigmatic process of restructuring into the primordial pre-40 subunit. We elucidate this process by biochemical and cryo-electron microscopy analysis of intermediates along this pathway in yeast. First, the remodeling RNA helicase Dhr1 engages the 90 pre-ribosome, followed by Utp24 endonuclease-driven RNA cleavage at site A, thereby separating the 5'-external transcribed spacer (ETS) from 18 ribosomal RNA. Next, the 5'-ETS and 90 assembly factors become dislodged, but this occurs sequentially, not en bloc. Eventually, the primordial pre-40 emerges, still retaining some 90 factors including Dhr1, now ready to unwind the final small nucleolar U3-18 RNA hybrid. Our data shed light on the elusive 90 to pre-40 transition and clarify the principles of assembly and remodeling of large ribonucleoproteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.abb4119DOI Listing
September 2020

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

PLoS Biol 2020 07 20;18(7):e3000780. Epub 2020 Jul 20.

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Cells adjust to nutrient deprivation by reversible translational shutdown. This is accompanied by maintaining inactive ribosomes in a hibernation state, in which they are bound by proteins with inhibitory and protective functions. In eukaryotes, such a function was attributed to suppressor of target of Myb protein 1 (Stm1; SERPINE1 mRNA-binding protein 1 [SERBP1] in mammals), and recently, late-annotated short open reading frame 2 (Lso2; coiled-coil domain containing short open reading frame 124 [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 nonrotated 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 (GAC) of the large subunit. This position allows accommodation of the duplication of multilocus region 34 protein (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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.3000780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392345PMC
July 2020

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

Science 2020 09 17;369(6508):1249-1255. Epub 2020 Jul 17.

Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40 ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo-electron microscopy of in vitro-reconstituted Nsp1-40 and various native Nsp1-40 and -80 complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid-inducible gene 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.abc8665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402621PMC
September 2020

Construction of the Central Protuberance and L1 Stalk during 60S Subunit Biogenesis.

Mol Cell 2020 08 14;79(4):615-628.e5. Epub 2020 Jul 14.

Biochemie-Zentrum der Universität Heidelberg, 69120 Heidelberg, Germany. Electronic address:

Ribosome assembly is driven by numerous assembly factors, including the Rix1 complex and the AAA ATPase Rea1. These two assembly factors catalyze 60S maturation at two distinct states, triggering poorly understood large-scale structural transitions that we analyzed by cryo-electron microscopy. Two nuclear pre-60S intermediates were discovered that represent previously unknown states after Rea1-mediated removal of the Ytm1-Erb1 complex and reveal how the L1 stalk develops from a pre-mature nucleolar to a mature-like nucleoplasmic state. A later pre-60S intermediate shows how the central protuberance arises, assisted by the nearby Rix1-Rea1 machinery, which was solved in its pre-ribosomal context to molecular resolution. This revealed a Rix1-Ipi3 tetramer anchored to the pre-60S via Ipi1, strategically positioned to monitor this decisive remodeling. These results are consistent with a general underlying principle that temporarily stabilized immature RNA domains are successively remodeled by assembly factors, thereby ensuring failsafe assembly progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.06.032DOI Listing
August 2020

Author Correction: 3.2-Å-resolution structure of the 90S preribosome before A1 pre-rRNA cleavage.

Nat Struct Mol Biol 2020 Jul;27(7):683

Department of Biochemistry, Gene Center Munich and Center of Integrated Protein Science-Munich (CiPS-M), University of Munich, Munich, Germany.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41594-020-0456-yDOI Listing
July 2020

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

Science 2020 04;368(6488)

Gene Center and Department of Biochemistry, University of Munich, 81377 Munich, Germany.

Control of messenger RNA (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. We used a combination of cryo-electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aay6912DOI Listing
April 2020

Molecular analysis of the ribosome recycling factor ABCE1 bound to the 30S post-splitting complex.

EMBO J 2020 05 17;39(9):e103788. Epub 2020 Feb 17.

Department of Biochemistry, Gene Center, Ludwig-Maximilians University Munich, München, Germany.

Ribosome recycling by the twin-ATPase ABCE1 is a key regulatory process in mRNA translation and surveillance and in ribosome-associated protein quality control in Eukarya and Archaea. Here, we captured the archaeal 30S ribosome post-splitting complex at 2.8 Å resolution by cryo-electron microscopy. The structure reveals the dynamic behavior of structural motifs unique to ABCE1, which ultimately leads to ribosome splitting. More specifically, we provide molecular details on how conformational rearrangements of the iron-sulfur cluster domain and hinge regions of ABCE1 are linked to closure of its nucleotide-binding sites. The combination of mutational and functional analyses uncovers an intricate allosteric network between the ribosome, regulatory domains of ABCE1, and its two structurally and functionally asymmetric ATP-binding sites. Based on these data, we propose a refined model of how signals from the ribosome are integrated into the ATPase cycle of ABCE1 to orchestrate ribosome recycling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.2019103788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196836PMC
May 2020

Structure of the Bcs1 AAA-ATPase suggests an airlock-like translocation mechanism for folded proteins.

Nat Struct Mol Biol 2020 02 27;27(2):142-149. Epub 2020 Jan 27.

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Some proteins require completion of folding before translocation across a membrane into another cellular compartment. Yet the permeability barrier of the membrane should not be compromised and mechanisms have remained mostly elusive. Here, we present the structure of Saccharomyces cerevisiae Bcs1, an AAA-ATPase of the inner mitochondrial membrane. Bcs1 facilitates the translocation of the Rieske protein, Rip1, which requires folding and incorporation of a 2Fe-2S cluster before translocation and subsequent integration into the bc1 complex. Surprisingly, Bcs1 assembles into exclusively heptameric homo-oligomers, with each protomer consisting of an amphipathic transmembrane helix, a middle domain and an ATPase domain. Together they form two aqueous vestibules, the first being accessible from the mitochondrial matrix and the second positioned in the inner membrane, with both separated by the seal-forming middle domain. On the basis of this unique architecture, we propose an airlock-like translocation mechanism for folded Rip1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41594-019-0364-1DOI Listing
February 2020

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

EMBO J 2020 02 20;39(3):e103365. Epub 2019 Dec 20.

Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

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- and collision-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 a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.2019103365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996574PMC
February 2020

Partially inserted nascent chain unzips the lateral gate of the Sec translocon.

EMBO Rep 2019 10 5;20(10):e48191. Epub 2019 Aug 5.

Gene Center Munich, Ludwig-Maximilian-University, Munich, Germany.

The Sec translocon provides the lipid bilayer entry for ribosome-bound nascent chains and thus facilitates membrane protein biogenesis. Despite the appreciated role of the native environment in the translocon:ribosome assembly, structural information on the complex in the lipid membrane is scarce. Here, we present a cryo-electron microscopy-based structure of bacterial translocon SecYEG in lipid nanodiscs and elucidate an early intermediate state upon insertion of the FtsQ anchor domain. Insertion of the short nascent chain causes initial displacements within the lateral gate of the translocon, where α-helices 2b, 7, and 8 tilt within the membrane core to "unzip" the gate at the cytoplasmic side. Molecular dynamics simulations demonstrate that the conformational change is reversed in the absence of the ribosome, and suggest that the accessory α-helices of SecE subunit modulate the lateral gate conformation. Site-specific cross-linking validates that the FtsQ nascent chain passes the lateral gate upon insertion. The structure and the biochemical data suggest that the partially inserted nascent chain remains highly flexible until it acquires the transmembrane topology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embr.201948191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776908PMC
October 2019

Thermophile 90S Pre-ribosome Structures Reveal the Reverse Order of Co-transcriptional 18S rRNA Subdomain Integration.

Mol Cell 2019 09 1;75(6):1256-1269.e7. Epub 2019 Aug 1.

Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. Electronic address:

Eukaryotic ribosome biogenesis involves RNA folding and processing that depend on assembly factors and small nucleolar RNAs (snoRNAs). The 90S (SSU-processome) is the earliest pre-ribosome structurally analyzed, which was suggested to assemble stepwise along the growing pre-rRNA from 5' > 3', but this directionality may not be accurate. Here, by analyzing the structure of a series of 90S assembly intermediates from Chaetomium thermophilum, we discover a reverse order of 18S rRNA subdomain incorporation. Large parts of the 18S rRNA 3' and central domains assemble first into the 90S before the 5' domain is integrated. This final incorporation depends on a contact between a heterotrimer Enp2-Bfr2-Lcp5 recruited to the flexible 5' domain and Kre33, which reconstitutes the Kre33-Enp-Brf2-Lcp5 module on the compacted 90S. Keeping the 5' domain temporarily segregated from the 90S scaffold could provide extra time to complete the multifaceted 5' domain folding, which depends on a distinct set of snoRNAs and processing factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2019.06.032DOI Listing
September 2019

Structural and mutational analysis of the ribosome-arresting human XBP1u.

Elife 2019 06 27;8. Epub 2019 Jun 27.

Gene Center, Department of Biochemistry, Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Munich, Germany.

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 Å cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the ribosomal exit tunnel proximal to the peptidyl transferase center where it causes a subtle distortion, thereby 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 8 out of 20 mutagenized positions are optimal; in the remaining 12 positions, we identify 55 different mutations increase the level of translational arrest. Thus, the wildtype XBP1u AP induces only an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.46267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624018PMC
June 2019

Publisher Correction: Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis.

Nature 2019 Jul;571(7764):E4

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Change history: In this Letter, the bottom blot in Fig. 2g (for 'IB: Myc') was missing. This has been corrected online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1360-7DOI Listing
July 2019

Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis.

Nature 2019 06 12;570(7762):538-542. Epub 2019 Jun 12.

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Ribosome-associated quality control (RQC) provides a rescue pathway for eukaryotic cells to process faulty proteins after translational stalling of cytoplasmic ribosomes. After dissociation of ribosomes, the stalled tRNA-bound peptide remains associated with the 60S subunit and extended by Rqc2 by addition of C-terminal alanyl and threonyl residues (CAT tails), whereas Vms1 catalyses cleavage and release of the peptidyl-tRNA before or after addition of CAT tails. In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis. Here we present structural and functional insights into the interaction of Saccharomyces cerevisiae Vms1 with 60S subunits in pre- and post-peptidyl-tRNA cleavage states. Vms1 binds to 60S subunits with its Vms1-like release factor 1 (VLRF1), zinc finger and ankyrin domains. VLRF1 overlaps with the Rqc2 A-tRNA position and interacts with the ribosomal A-site, projecting its catalytic GSQ motif towards the CCA end of the tRNA, its Y285 residue dislodging the tRNA A73 for nucleolytic cleavage. Moreover, in the pre-state, we found the ABCF-type ATPase Arb1 in the ribosomal E-site, which stabilizes the delocalized A73 of the peptidyl-tRNA and stimulates Vms1-dependent tRNA cleavage. Our structural analysis provides mechanistic insights into the interplay of the RQC factors Vms1, Rqc2 and Arb1 and their role in the protection of mitochondria from the aggregation of toxic proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1307-zDOI Listing
June 2019

Structure of the 80S ribosome-Xrn1 nuclease complex.

Nat Struct Mol Biol 2019 04 25;26(4):275-280. Epub 2019 Mar 25.

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Messenger RNA (mRNA) homeostasis represents an essential part of gene expression, in which the generation of mRNA by RNA polymerase is counter-balanced by its degradation by nucleases. The conserved 5'-to-3' exoribonuclease Xrn1 has a crucial role in eukaryotic mRNA homeostasis by degrading decapped or cleaved mRNAs post-translationally and, more surprisingly, also co-translationally. Here we report that active Xrn1 can directly and specifically interact with the translation machinery. A cryo-electron microscopy structure of a programmed Saccharomyces cerevisiae 80S ribosome-Xrn1 nuclease complex reveals how the conserved core of Xrn1 enables binding at the mRNA exit site of the ribosome. This interface provides a conduit for channelling of the mRNA from the ribosomal decoding site directly into the active center of the nuclease, thus separating mRNA decoding from degradation by only 17 ± 1 nucleotides. These findings explain how rapid 5'-to-3' mRNA degradation is coupled efficiently to its final round of mRNA translation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41594-019-0202-5DOI Listing
April 2019

Ribosome-NatA architecture reveals that rRNA expansion segments coordinate N-terminal acetylation.

Nat Struct Mol Biol 2019 01 17;26(1):35-39. Epub 2018 Dec 17.

Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

The majority of eukaryotic proteins are N-terminally α-acetylated by N-terminal acetyltransferases (NATs). Acetylation usually occurs co-translationally and defects have severe consequences. Nevertheless, it is unclear how these enzymes act in concert with the translating ribosome. Here, we report the structure of a native ribosome-NatA complex from Saccharomyces cerevisiae. NatA (comprising Naa10, Naa15 and Naa50) displays a unique mode of ribosome interaction by contacting eukaryotic-specific ribosomal RNA expansion segments in three out of four binding patches. Thereby, NatA is dynamically positioned directly underneath the ribosomal exit tunnel to facilitate modification of the emerging nascent peptide chain. Methionine amino peptidases, but not chaperones or signal recognition particle, would be able to bind concomitantly. This work assigns a function to the hitherto enigmatic ribosomal RNA expansion segments and provides mechanistic insights into co-translational protein maturation by N-terminal acetylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41594-018-0165-yDOI Listing
January 2019

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

Nat Microbiol 2018 10 3;3(10):1115-1121. Epub 2018 Sep 3.

Institute for Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany.

To survive under conditions of stress, such as nutrient deprivation, bacterial 70S ribosomes dimerize to form hibernating 100S particles. In γ-proteobacteria, such as Escherichia coli, 100S formation requires the ribosome modulation factor (RMF) and the hibernation promoting factor (HPF). Here we present single-particle cryo-electron microscopy structures of hibernating 70S and 100S particles isolated from stationary-phase E. coli cells at 3.0 Å and 7.9 Å resolution, respectively. The structures reveal the binding sites for HPF and RMF as well as the unexpected presence of deacylated E-site transfer RNA and ribosomal protein bS1. HPF interacts with the anticodon-stem-loop of the E-tRNA and occludes the binding site for the messenger RNA as well as A- and P-site tRNAs. RMF facilitates stabilization of a compact conformation of bS1, which together sequester the anti-Shine-Dalgarno sequence of the 16S ribosomal RNA (rRNA), thereby inhibiting translation initiation. At the dimerization interface, the C-terminus of uS2 probes the mRNA entrance channel of the symmetry-related particle, thus suggesting that dimerization inactivates ribosomes by blocking the binding of mRNA within the channel. The back-to-back E. coli 100S arrangement is distinct from 100S particles observed previously in Gram-positive bacteria, and reveals a unique role for bS1 in translation regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-018-0237-0DOI Listing
October 2018

Visualizing late states of human 40S ribosomal subunit maturation.

Nature 2018 06 6;558(7709):249-253. Epub 2018 Jun 6.

Gene Center Munich and Center of Integrated Protein Science-Munich (CiPS-M), Department of Biochemistry, University of Munich, Munich, Germany.

The formation of eukaryotic ribosomal subunits extends from the nucleolus to the cytoplasm and entails hundreds of assembly factors. Despite differences in the pathways of ribosome formation, high-resolution structural information has been available only from fungi. Here we present cryo-electron microscopy structures of late-stage human 40S assembly intermediates, representing one state reconstituted in vitro and five native states that range from nuclear to late cytoplasmic. The earliest particles reveal the position of the biogenesis factor RRP12 and distinct immature rRNA conformations that accompany the formation of the 40S subunit head. Molecular models of the late-acting assembly factors TSR1, RIOK1, RIOK2, ENP1, LTV1, PNO1 and NOB1 provide mechanistic details that underlie their contribution to a sequential 40S subunit assembly. The NOB1 architecture displays an inactive nuclease conformation that requires rearrangement of the PNO1-bound 3' rRNA, thereby coordinating the final rRNA folding steps with site 3 cleavage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0193-0DOI Listing
June 2018

Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum.

Science 2018 04 8;360(6385):215-219. Epub 2018 Mar 8.

Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich, University of Munich, 81377 Munich, Germany.

Protein synthesis, transport, and N-glycosylation are coupled at the mammalian endoplasmic reticulum by complex formation of a ribosome, the Sec61 protein-conducting channel, and oligosaccharyltransferase (OST). Here we used different cryo-electron microscopy approaches to determine structures of native and solubilized ribosome-Sec61-OST complexes. A molecular model for the catalytic OST subunit STT3A (staurosporine and temperature sensitive 3A) revealed how it is integrated into the OST and how STT3-paralog specificity for translocon-associated OST is achieved. The OST subunit DC2 was placed at the interface between Sec61 and STT3A, where it acts as a versatile module for recruitment of STT3A-containing OST to the ribosome-Sec61 complex. This detailed structural view on the molecular architecture of the cotranslational machinery for N-glycosylation provides the basis for a mechanistic understanding of glycoprotein biogenesis at the endoplasmic reticulum.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aar7899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319373PMC
April 2018

Visualizing the Assembly Pathway of Nucleolar Pre-60S Ribosomes.

Cell 2017 Dec;171(7):1599-1610.e14

Gene Center Munich and Center of Integrated Protein Science-Munich (CiPS-M), Department of Biochemistry, Feodor-Lynen-Str. 25, University of Munich, 81377 Munich, Germany. Electronic address:

Eukaryotic 60S ribosomal subunits are comprised of three rRNAs and ∼50 ribosomal proteins. The initial steps of their formation take place in the nucleolus, but, owing to a lack of structural information, this process is poorly understood. Using cryo-EM, we solved structures of early 60S biogenesis intermediates at 3.3 Å to 4.5 Å resolution, thereby providing insights into their sequential folding and assembly pathway. Besides revealing distinct immature rRNA conformations, we map 25 assembly factors in six different assembly states. Notably, the Nsa1-Rrp1-Rpf1-Mak16 module stabilizes the solvent side of the 60S subunit, and the Erb1-Ytm1-Nop7 complex organizes and connects through Erb1's meandering N-terminal extension, eight assembly factors, three ribosomal proteins, and three 25S rRNA domains. Our structural snapshots reveal the order of integration and compaction of the six major 60S domains within early nucleolar 60S particles developing stepwise from the solvent side around the exit tunnel to the central protuberance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2017.11.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5745149PMC
December 2017

Cryo-EM structure of a late pre-40S ribosomal subunit from .

Elife 2017 11 20;6. Epub 2017 Nov 20.

Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Mechanistic understanding of eukaryotic ribosome formation requires a detailed structural knowledge of the numerous assembly intermediates, generated along a complex pathway. Here, we present the structure of a late pre-40S particle at 3.6 Å resolution, revealing in molecular detail how assembly factors regulate the timely folding of pre-18S rRNA. The structure shows that, rather than sterically blocking 40S translational active sites, the associated assembly factors Tsr1, Enp1, Rio2 and Pno1 collectively preclude their final maturation, thereby preventing untimely tRNA and mRNA binding and error prone translation. Moreover, the structure explains how Pno1 coordinates the 3'end cleavage of the 18S rRNA by Nob1 and how the late factor's removal in the cytoplasm ensures the structural integrity of the maturing 40S subunit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.30189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695908PMC
November 2017

Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P.

Mol Cell 2017 Nov;68(3):515-527.e6

Gene Center, Department for Biochemistry and Center for integrated Protein Science Munich (CiPSM), University of Munich, Feodor-Lynenstr. 25, 81377 Munich, Germany; Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany. Electronic address:

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2017.10.014DOI Listing
November 2017

3.2-Å-resolution structure of the 90S preribosome before A1 pre-rRNA cleavage.

Nat Struct Mol Biol 2017 Nov 2;24(11):954-964. Epub 2017 Oct 2.

Gene Center Munich and Center of Integrated Protein Science-Munich (CiPS-M), Department of Biochemistry, University of Munich, Munich, Germany.

The 40S small ribosomal subunit is cotranscriptionally assembled in the nucleolus as part of a large chaperone complex called the 90S preribosome or small-subunit processome. Here, we present the 3.2-Å-resolution structure of the Chaetomium thermophilum 90S preribosome, which allowed us to build atomic structures for 34 assembly factors, including the Mpp10 complex, Bms1, Utp14 and Utp18, and the complete U3 small nucleolar ribonucleoprotein. Moreover, we visualized the U3 RNA heteroduplexes with a 5' external transcribed spacer (5' ETS) and pre-18S RNA, and their stabilization by 90S factors. Overall, the structure explains how a highly intertwined network of assembly factors and pre-rRNA guide the sequential, independent folding of the individual pre-40S domains while the RNA regions forming the 40S active sites are kept immature. Finally, by identifying the unprocessed A1 cleavage site and the nearby Utp24 endonuclease, we suggest a proofreading model for regulated 5'-ETS separation and 90S-pre-40S transition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.3476DOI Listing
November 2017

An antimicrobial peptide that inhibits translation by trapping release factors on the ribosome.

Nat Struct Mol Biol 2017 Sep 24;24(9):752-757. Epub 2017 Jul 24.

Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, Illinois, USA.

Many antibiotics stop bacterial growth by inhibiting different steps of protein synthesis. However, no specific inhibitors of translation termination are known. Proline-rich antimicrobial peptides, a component of the antibacterial defense system of multicellular organisms, interfere with bacterial growth by inhibiting translation. Here we show that Api137, a derivative of the insect-produced antimicrobial peptide apidaecin, arrests terminating ribosomes using a unique mechanism of action. Api137 binds to the Escherichia coli ribosome and traps release factor (RF) RF1 or RF2 subsequent to the release of the nascent polypeptide chain. A high-resolution cryo-EM structure of the ribosome complexed with RF1 and Api137 reveals the molecular interactions that lead to RF trapping. Api137-mediated depletion of the cellular pool of free release factors causes the majority of ribosomes to stall at stop codons before polypeptide release, thereby resulting in a global shutdown of translation termination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.3439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589491PMC
September 2017

The force-sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling.

Elife 2017 05 30;6. Epub 2017 May 30.

Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich, Ludwig Maximilian University of Munich, Munich, Germany.

Interaction between the nascent polypeptide chain and the ribosomal exit tunnel can modulate the rate of translation and induce translational arrest to regulate expression of downstream genes. The ribosomal tunnel also provides a protected environment for initial protein folding events. Here, we present a 2.9 Å cryo-electron microscopy structure of a ribosome stalled during translation of the extremely compacted VemP nascent chain. The nascent chain forms two α-helices connected by an α-turn and a loop, enabling a total of 37 amino acids to be observed within the first 50-55 Å of the exit tunnel. The structure reveals how α-helix formation directly within the peptidyltransferase center of the ribosome interferes with aminoacyl-tRNA accommodation, suggesting that during canonical translation, a major role of the exit tunnel is to prevent excessive secondary structure formation that can interfere with the peptidyltransferase activity of the ribosome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.25642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449182PMC
May 2017