ABSTRACT
Eukaryotic 60S ribosomal subunits are comprised of three rRNAs and â¼50 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.
Subject(s)
Chaetomium/chemistry , Organelle Biogenesis , Ribosome Subunits, Large, Eukaryotic/chemistry , Chaetomium/cytology , Cryoelectron Microscopy , Metabolic Networks and Pathways , Models, Molecular , RNA Folding , Ribonucleoproteins/chemistryABSTRACT
The 90S pre-ribosome is an early biogenesis intermediate formed during co-transcriptional ribosome formation, composed of â¼70 assembly factors and several small nucleolar RNAs (snoRNAs) that associate with nascent pre-rRNA. We report the cryo-EM structure of the Chaetomium thermophilum 90S pre-ribosome, revealing how a network of biogenesis factors including 19 ß-propellers and large α-solenoid proteins engulfs the pre-rRNA. Within the 90S pre-ribosome, we identify the UTP-A, UTP-B, Mpp10-Imp3-Imp4, Bms1-Rcl1, and U3 snoRNP modules, which are organized around 5'-ETS and partially folded 18S rRNA. The U3 snoRNP is strategically positioned at the center of the 90S particle to perform its multiple tasks during pre-rRNA folding and processing. The architecture of the elusive 90S pre-ribosome gives unprecedented structural insight into the early steps of pre-rRNA maturation. Nascent rRNA that is co-transcriptionally folded and given a particular shape by encapsulation within a dedicated mold-like structure is reminiscent of how polypeptides use chaperone chambers for their protein folding.
Subject(s)
Chaetomium/chemistry , Organelle Biogenesis , Ribosomes/chemistry , Saccharomyces cerevisiae/chemistry , Chaetomium/classification , Cryoelectron Microscopy , Models, Molecular , RNA, Ribosomal, 18S/chemistry , Ribosome Subunits, Large, Eukaryotic/chemistry , Ribosome Subunits, Small, Eukaryotic/chemistry , Ribosomes/ultrastructureABSTRACT
Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on noncolliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S nonfunctional rRNA decay via polyubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also an association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting an mRNA stasis sensing activity, and Fap1 sterically hinders the formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.
Subject(s)
Protein Biosynthesis , Ribosomes , RNA Stability , RNA, Messenger/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Ribosomes/metabolismABSTRACT
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, A1, 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.
Subject(s)
DEAD-box RNA Helicases/chemistry , Endoribonucleases/chemistry , Exonucleases/chemistry , Exosome Multienzyme Ribonuclease Complex/ultrastructure , RNA, Ribosomal, 18S/chemistry , Ribosomes/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/genetics , Binding Sites , Cryoelectron Microscopy , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Endoribonucleases/genetics , Endoribonucleases/metabolism , Exonucleases/genetics , Exonucleases/metabolism , Exosome Multienzyme Ribonuclease Complex/genetics , Exosome Multienzyme Ribonuclease Complex/metabolism , Models, Molecular , Protein Binding , Protein Biosynthesis , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA Stability , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 18S/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
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 the 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 we 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 through the ribosome quality control pathway. However, unlike SmrB, which cleaves mRNA in E. coli, we see no evidence that MutS2 mediates mRNA cleavage or promotes ribosome rescue by tmRNA. 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 diverse bacteria.
Subject(s)
Bacillus subtilis , Protein Biosynthesis , RNA, Messenger/metabolism , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Ribosomes/metabolism , Peptides/metabolismABSTRACT
INO80/SWR1 family chromatin remodelers are complexes composed of >15 subunits and molecular masses exceeding 1 MDa. Their important role in transcription and genome maintenance is exchanging the histone variants H2A and H2A.Z. We report the architecture of S. cerevisiae INO80 using an integrative approach of electron microscopy, crosslinking and mass spectrometry. INO80 has an embryo-shaped head-neck-body-foot architecture and shows dynamic open and closed conformations. We can assign an Rvb1/Rvb2 heterododecamer to the head in close contact with the Ino80 Snf2 domain, Ies2, and the Arp5 module at the neck. The high-affinity nucleosome-binding Nhp10 module localizes to the body, whereas the module that contains actin, Arp4, and Arp8 maps to the foot. Structural and biochemical analyses indicate that the nucleosome is bound at the concave surface near the neck, flanked by the Rvb1/2 and Arp8 modules. Our analysis establishes a structural and functional framework for this family of large remodelers.
Subject(s)
Nucleosomes/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/metabolism , Chromatin Assembly and Disassembly , Mass Spectrometry , Models, Molecular , Nucleosomes/metabolism , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/ultrastructure , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation1,2. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes3-6. Here, using a genetic screen in Escherichia coli, we discovered a new rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNAs3) to rescue upstream ribosomes. SmrB is recruited to ribosomes and is activated by collisions. Cryo-electron microscopy structures of collided disomes from E. coli and Bacillus subtilis show distinct and conserved arrangements of individual ribosomes and the composite SmrB-binding site. These findings reveal the underlying mechanisms by which ribosome collisions trigger ribosome rescue in bacteria.
Subject(s)
Escherichia coli , Ribosomes , Bacteria/genetics , Cryoelectron Microscopy , Escherichia coli/genetics , Escherichia coli/metabolism , Protein Biosynthesis , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , Ribosomes/metabolismABSTRACT
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 Rix12-Ipi32 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.
Subject(s)
Ribosome Subunits, Large, Eukaryotic/chemistry , Ribosome Subunits, Large, Eukaryotic/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , ATPases Associated with Diverse Cellular Activities/metabolism , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Cryoelectron Microscopy , Escherichia coli/genetics , Models, Molecular , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
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.
Subject(s)
Chaetomium/metabolism , Fungal Proteins/metabolism , Nucleic Acid Conformation , RNA, Fungal/metabolism , RNA, Ribosomal, 18S/metabolism , Ribosomes/metabolism , Chaetomium/genetics , Fungal Proteins/genetics , RNA, Fungal/genetics , RNA, Ribosomal, 18S/genetics , Ribosomes/geneticsABSTRACT
Cotranslational modification of the nascent polypeptide chain is one of the first events during the birth of a new protein. In eukaryotes, methionine aminopeptidases (MetAPs) cleave off the starter methionine, whereas N-acetyl-transferases (NATs) catalyze N-terminal acetylation. MetAPs and NATs compete with other cotranslationally acting chaperones, such as ribosome-associated complex (RAC), protein targeting and translocation factors (SRP and Sec61) for binding sites at the ribosomal tunnel exit. Yet, whereas well-resolved structures for ribosome-bound RAC, SRP and Sec61, are available, structural information on the mode of ribosome interaction of eukaryotic MetAPs or of the five cotranslationally active NATs is only available for NatA. Here, we present cryo-EM structures of yeast Map1 and NatB bound to ribosome-nascent chain complexes. Map1 is mainly associated with the dynamic rRNA expansion segment ES27a, thereby kept at an ideal position below the tunnel exit to act on the emerging substrate nascent chain. For NatB, we observe two copies of the NatB complex. NatB-1 binds directly below the tunnel exit, again involving ES27a, and NatB-2 is located below the second universal adapter site (eL31 and uL22). The binding mode of the two NatB complexes on the ribosome differs but overlaps with that of NatA and Map1, implying that NatB binds exclusively to the tunnel exit. We further observe that ES27a adopts distinct conformations when bound to NatA, NatB, or Map1, together suggesting a contribution to the coordination of a sequential activity of these factors on the emerging nascent chain at the ribosomal exit tunnel.
Subject(s)
Peptides , Ribosomes , Ribosomes/metabolism , Peptides/chemistry , RNA, Ribosomal/metabolism , Binding Sites , Saccharomyces cerevisiae/genetics , Methionine/metabolism , Protein Biosynthesis , Acetyltransferases/analysis , Acetyltransferases/genetics , Acetyltransferases/metabolismABSTRACT
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 proteins1,2. Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3 by the endonuclease NOB13. Previous structures of human 40S precursors have shown that NOB1 is kept in an inactive state by its partner PNO14. 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.
Subject(s)
Cryoelectron Microscopy , Ribosome Subunits, Small, Eukaryotic/chemistry , Ribosome Subunits, Small, Eukaryotic/metabolism , Enzyme Activation , HeLa Cells , Humans , Models, Molecular , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nuclear Proteins/ultrastructure , Protein Conformation , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/ultrastructure , Proteins/chemistry , Proteins/metabolism , Proteins/ultrastructure , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/ultrastructure , Ribosome Subunits, Small, Eukaryotic/ultrastructure , Saccharomyces cerevisiae/chemistryABSTRACT
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.
Subject(s)
Heat-Shock Proteins/chemistry , Heat-Shock Proteins/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Binding Sites , Cryoelectron Microscopy , Crystallography, X-Ray , Endoplasmic Reticulum/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Protein Domains , Protein Processing, Post-Translational , Saccharomyces cerevisiae/chemistryABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Cell Line , DNA-Binding Proteins/metabolism , Eukaryotic Initiation Factor-2/metabolism , HEK293 Cells , Humans , Protein Binding/physiology , Protein Biosynthesis/physiology , RNA, Messenger/metabolism , RNA, Transfer/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Ribosome biogenesis proceeds along a multifaceted pathway from the nucleolus to the cytoplasm that is extensively coupled to several quality control mechanisms. However, the mode by which 5S ribosomal RNA is incorporated into the developing pre-60S ribosome, which in humans links ribosome biogenesis to cell proliferation by surveillance by factors such as p53-MDM2, is poorly understood. Here, we report nine nucleolar pre-60S cryo-EM structures from Chaetomium thermophilum, one of which clarifies the mechanism of 5S RNP incorporation into the early pre-60S. Successive assembly states then represent how helicases Dbp10 and Spb4, and the Pumilio domain factor Puf6 act in series to surveil the gradual folding of the nearby 25S rRNA domain IV. Finally, the methyltransferase Spb1 methylates a universally conserved guanine nucleotide in the A-loop of the peptidyl transferase center, thereby licensing further maturation. Our findings provide insight into the hierarchical action of helicases in safeguarding rRNA tertiary structure folding and coupling to surveillance mechanisms that culminate in local RNA modification.
Subject(s)
RNA, Ribosomal , Saccharomyces cerevisiae Proteins , Humans , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Ribosomes/genetics , RNA, Ribosomal, 5S/genetics , RNA, Ribosomal, 5S/metabolism , DNA Helicases/metabolism , Protein Binding , Ribosomal Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The rixosome defined in Schizosaccharomyces pombe and humans performs diverse roles in pre-ribosomal RNA processing and gene silencing. Here, we isolate and describe the conserved rixosome from Chaetomium thermophilum, which consists of two sub-modules, the sphere-like Rix1-Ipi3-Ipi1 and the butterfly-like Las1-Grc3 complex, connected by a flexible linker. The Rix1 complex of the rixosome utilizes Sda1 as landing platform on nucleoplasmic pre-60S particles to wedge between the 5S rRNA tip and L1-stalk, thereby facilitating the 180° rotation of the immature 5S RNP towards its mature conformation. Upon rixosome positioning, the other sub-module with Las1 endonuclease and Grc3 polynucleotide-kinase can reach a strategic position at the pre-60S foot to cleave and 5' phosphorylate the nearby ITS2 pre-rRNA. Finally, inward movement of the L1 stalk permits the flexible Nop53 N-terminus with its AIM motif to become positioned at the base of the L1-stalk to facilitate Mtr4 helicase-exosome participation for completing ITS2 removal. Thus, the rixosome structure elucidates the coordination of two central ribosome biogenesis events, but its role in gene silencing may adapt similar strategies.
Subject(s)
Saccharomyces cerevisiae Proteins , Schizosaccharomyces , Humans , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Nuclear Proteins/metabolism , Rotation , RNA, Ribosomal/metabolism , Ribosomes/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , RNA Processing, Post-Transcriptional , Ribosomal Proteins/geneticsABSTRACT
RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal structure of Maf1 and cryo-electron microscopic structures of Pol III, an active Pol III-DNA-RNA complex, and a repressive Pol III-Maf1 complex. Binding of DNA and RNA causes ordering of the Pol III-specific subcomplex C82/34/31 that is required for transcription initiation. Maf1 binds the Pol III clamp and rearranges C82/34/31 at the rim of the active center cleft. This impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors Brf1 and TBP and thus prevents closed complex formation. Maf1 does however not impair binding of a DNA-RNA scaffold and RNA synthesis. These results explain how Maf1 specifically represses transcription initiation from Pol III promoters and indicate that Maf1 also prevents reinitiation by binding Pol III during transcription elongation.
Subject(s)
RNA Polymerase III/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Molecular Sequence Data , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Promoter Regions, Genetic , RNA Polymerase III/antagonists & inhibitors , RNA Polymerase III/chemistry , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , Transcription Factors/chemistry , Transcription, GeneticABSTRACT
Change history: In this Letter, the bottom blot in Fig. 2g (for 'IB: Myc') was missing. This has been corrected online.
ABSTRACT
Ribosome-associated quality control (RQC) provides a rescue pathway for eukaryotic cells to process faulty proteins after translational stalling of cytoplasmic ribosomes1-6. 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)7-9, whereas Vms1 catalyses cleavage and release of the peptidyl-tRNA before or after addition of CAT tails10-12. In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis13. 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.
Subject(s)
ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Homeostasis , Mitochondrial Proteins/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/ultrastructure , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/ultrastructure , Amino Acid Sequence , Carrier Proteins/ultrastructure , Cryoelectron Microscopy , Models, Molecular , Proteome/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/chemistry , Ribosome Subunits, Large, Eukaryotic/genetics , Ribosome Subunits, Large, Eukaryotic/metabolism , Ribosomes/chemistry , Ribosomes/genetics , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/ultrastructureABSTRACT
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.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Peptide Elongation Factors/metabolism , Peptides/metabolism , Ribosomes/metabolism , Binding Sites , Cryoelectron Microscopy , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/ultrastructure , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Nucleic Acid Conformation , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/genetics , Peptide Elongation Factors/ultrastructure , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/metabolism , Peptides/chemistry , Protein Binding , Protein Biosynthesis , Protein Conformation , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer/chemistry , RNA, Transfer/genetics , RNA, Transfer/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Ribosomes/chemistry , Ribosomes/ultrastructure , Structure-Activity Relationship , Eukaryotic Translation Initiation Factor 5AABSTRACT
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.