ABSTRACT
Nonsense mutations account for >10% of human genetic disorders, including cystic fibrosis, Alagille syndrome, and Duchenne muscular dystrophy. A nonsense mutation results in the expression of a truncated protein, and therapeutic strategies aim to restore full-length protein expression. Most strategies under development, including small-molecule aminoglycosides, suppressor tRNAs, or the targeted degradation of termination factors, lack mRNA target selectivity and may poorly differentiate between nonsense and normal stop codons, resulting in off-target translation errors. Here, we demonstrate that antisense oligonucleotides can stimulate readthrough of disease-causing nonsense codons, resulting in high yields of full-length protein in mammalian cellular lysate. Readthrough efficiency depends on the sequence context near the stop codon and on the precise targeting position of an oligonucleotide, whose interaction with mRNA inhibits peptide release to promote readthrough. Readthrough-inducing antisense oligonucleotides (R-ASOs) enhance the potency of non-specific readthrough agents, including aminoglycoside G418 and suppressor tRNA, enabling a path toward target-specific readthrough of nonsense mutations in CFTR, JAG1, DMD, BRCA1 and other mutant genes. Finally, through systematic chemical engineering, we identify heavily modified fully functional R-ASO variants, enabling future therapeutic development.
Subject(s)
Codon, Nonsense , Cystic Fibrosis Transmembrane Conductance Regulator , Oligonucleotides, Antisense , RNA, Messenger , Codon, Nonsense/genetics , Oligonucleotides, Antisense/genetics , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Protein Biosynthesis/drug effects , RNA, Transfer/genetics , RNA, Transfer/metabolism , Dystrophin/genetics , HEK293 Cells , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/therapy , Cystic Fibrosis/genetics , Cystic Fibrosis/drug therapy , GentamicinsABSTRACT
Angiogenin, an RNase-A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases and epigenetic inheritance1-10. After activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, resulting in translation repression11-15. However, the catalytic activity of isolated angiogenin is very low, and the mechanisms of the enzyme activation and tRNA specificity have remained a puzzle3,16-23. Here we identify these mechanisms using biochemical assays and cryogenic electron microscopy (cryo-EM). Our study reveals that the cytosolic ribosome is the activator of angiogenin. A cryo-EM structure features angiogenin bound in the A site of the 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the ribosome to activate the RNase catalytic centre, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Additional 80S-angiogenin structures capture how tRNA substrate is directed by the ribosome into angiogenin's active site, demonstrating that the ribosome acts as the specificity factor. Our findings therefore suggest that angiogenin is activated by ribosomes with a vacant A site, the abundance of which increases during cellular stress24-27. These results may facilitate the development of therapeutics to treat cancer and neurodegenerative diseases.
Subject(s)
Cryoelectron Microscopy , Ribonuclease, Pancreatic , Ribosomes , Humans , Anticodon/chemistry , Anticodon/genetics , Anticodon/metabolism , Anticodon/ultrastructure , Catalytic Domain , Cytosol/metabolism , Enzyme Activation , Models, Molecular , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/metabolism , Ribonuclease, Pancreatic/ultrastructure , Ribosomes/metabolism , Ribosomes/chemistry , Ribosomes/ultrastructure , RNA Cleavage , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Substrate Specificity , Binding Sites , Stress, PhysiologicalABSTRACT
GCN2 is a conserved receptor kinase activating the Integrated Stress Response (ISR) in eukaryotic cells. The ISR kinases detect accumulation of stress molecules and reprogram translation from basal tasks to preferred production of cytoprotective proteins. GCN2 stands out evolutionarily among all protein kinases due to the presence of a h istidyl t R NA s ynthetase-like (HRSL) domain, which arises only in GCN2 and is located next to the kinase domain. How HRSL contributes to GCN2 signaling remains unknown. Here we report a 3.2 Å cryo-EM structure of HRSL from thermotolerant yeast Kluyveromyces marxianus . This structure shows a constitutive symmetrical homodimer featuring a compact helical-bundle structure at the junction between HRSL and kinase domains, in the core of the receptor. Mutagenesis demonstrates that this junction structure activates GCN2 and indicates that our cryo-EM structure captures the active signaling state of HRSL. Based on these results, we put forward a GCN2 regulation mechanism, where HRSL drives the formation of activated kinase dimers. Remaining domains of GCN2 have the opposite role and in the absence of stress they help keep GCN2 basally inactive. This autoinhibitory activity is relieved upon stress ligand binding. We propose that the opposing action of HRSL and additional GCN2 domains thus yields a regulated ISR receptor. Significance statement: Regulation of protein synthesis (translation) is a central mechanism by which eukaryotic cells adapt to stressful conditions. In starving cells, this translational adaptation is achieved via the receptor kinase GCN2, which stays inactive under normal conditions, but is switched on under stress. The molecular mechanism of GCN2 switching is not well understood due to the presence of a structurally and biochemically uncharacterized h istidyl t R NA s ynthetase-like domain (HRSL) at the core of GCN2. Here we use single-particle cryo-EM and biochemistry to elucidate the structure and function of HRSL. We identify a structure at the kinase/HRSL interface, which forms crossed helices and helps position GCN2 kinase domains for activation. These data clarify the molecular mechanism of GCN2 regulation.
ABSTRACT
Ribosomes stall on truncated or otherwise damaged mRNAs. Bacteria rely on ribosome rescue mechanisms to replenish the pool of ribosomes available for translation. Trans-translation, the main ribosome-rescue pathway, uses a circular hybrid transfer-messenger RNA (tmRNA) to restart translation and label the resulting peptide for degradation. Previous studies have visualized how tmRNA and its helper protein SmpB interact with the stalled ribosome to establish a new open reading frame. As tmRNA presents the first alanine codon via a non-canonical mRNA path in the ribosome, the incoming alanyl-tRNA must rearrange the tmRNA molecule to read the codon. Here, we describe cryo-EM analyses of an endogenous Escherichia coli ribosome-tmRNA complex with tRNAAla accommodated in the A site. The flexible adenosine-rich tmRNA linker, which connects the mRNA-like domain with the codon, is stabilized by the minor groove of the canonically positioned anticodon stem of tRNAAla. This ribosome complex can also accommodate a tRNA near the E (exit) site, bringing insights into the translocation and dissociation of the tRNA that decoded the defective mRNA prior to tmRNA binding. Together, these structures uncover a key step of ribosome rescue, in which the ribosome starts translating the tmRNA reading frame.
ABSTRACT
In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner LoqsPB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer1â LoqsPB heterodimer. The Dicer-1 dsRBD and three LoqsPB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer1â LoqsPB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.
Subject(s)
Drosophila Proteins , MicroRNAs , Animals , Ribonuclease III/genetics , Ribonuclease III/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , RNA-Binding Proteins/metabolism , Drosophila/genetics , MicroRNAs/genetics , MicroRNAs/metabolismABSTRACT
Toxic dipeptide-repeat (DPR) proteins are produced from expanded G4C2 repeats in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥20 repeats inhibit the ribosome's peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryogenic electron microscopy (cryo-EM) reveals that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center (PTC). Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with poly-PR and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.
Subject(s)
Amyotrophic Lateral Sclerosis , Frontotemporal Dementia , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Cryoelectron Microscopy , Dipeptides/metabolism , Frontotemporal Dementia/genetics , Frontotemporal Dementia/metabolism , Humans , Proteins/genetics , Proteins/metabolism , Ribosomes/metabolism , TransferasesABSTRACT
Accurate protein synthesis (translation) relies on translation factors that rectify ribosome fluctuations into a unidirectional process. Understanding this process requires structural characterization of the ribosome and translation-factor dynamics. In the 2000s, crystallographic studies determined high-resolution structures of ribosomes stalled with translation factors, providing a starting point for visualizing translation. Recent progress in single-particle cryogenic electron microscopy (cryo-EM) has enabled near-atomic resolution of numerous structures sampled in heterogeneous complexes (ensembles). Ensemble and time-resolved cryo-EM have now revealed unprecedented views of ribosome transitions in the three principal stages of translation: initiation, elongation, and termination. This review focuses on how translation factors help achieve high accuracy and efficiency of translation by monitoring distinct ribosome conformations and by differentially shifting the equilibria of ribosome rearrangements for cognate and near-cognate substrates.
Subject(s)
Ribosomes , Single Molecule Imaging , Cryoelectron Microscopy , Ribosomes/metabolismABSTRACT
During translation, a conserved GTPase elongation factor-EF-G in bacteria or eEF2 in eukaryotes-translocates tRNA and mRNA through the ribosome. EF-G has been proposed to act as a flexible motor that propels tRNA and mRNA movement, as a rigid pawl that biases unidirectional translocation resulting from ribosome rearrangements, or by various combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive structures of ribosomeâ¢EF-G intermediates at near-atomic resolution. Prior to translocation, EF-G binds near peptidyl-tRNA, while the rotated 30S subunit stabilizes the EF-G GTPase center. Reverse 30S rotation releases Pi and translocates peptidyl-tRNA and EF-G by ~20 Å. An additional 4-Å translocation initiates EF-G dissociation from a transient ribosome state with highly swiveled 30S head. The structures visualize how nearly rigid EF-G rectifies inherent and spontaneous ribosomal dynamics into tRNA-mRNA translocation, whereas GTP hydrolysis and Pi release drive EF-G dissociation.
Subject(s)
Cryoelectron Microscopy , Guanosine Triphosphate/chemistry , Peptide Elongation Factor G/chemistry , Ribosomes/chemistry , Escherichia coli/chemistry , Escherichia coli/metabolism , Guanosine Triphosphate/metabolism , Peptide Elongation Factor G/metabolism , Phosphates/metabolism , Protein Binding , Protein Biosynthesis , RNA, Messenger/metabolism , RNA, Transfer/metabolism , RNA, Transfer, Amino Acyl/metabolism , Ribosome Subunits, Small, Bacterial/chemistry , Ribosome Subunits, Small, Bacterial/metabolism , Ribosomes/metabolismABSTRACT
When a ribosome encounters the stop codon of an mRNA, it terminates translation, releases the newly made protein, and is recycled to initiate translation on a new mRNA. Termination is a highly dynamic process in which release factors (RF1 and RF2 in bacteria; eRF1â¢eRF3â¢GTP in eukaryotes) coordinate peptide release with large-scale molecular rearrangements of the ribosome. Ribosomes stalled on aberrant mRNAs are rescued and recycled by diverse bacterial, mitochondrial, or cytoplasmic quality control mechanisms. These are catalyzed by rescue factors with peptidyl-tRNA hydrolase activity (bacterial ArfAâ¢RF2 and ArfB, mitochondrial ICT1 and mtRF-R, and cytoplasmic Vms1), that are distinct from each other and from release factors. Nevertheless, recent structural studies demonstrate a remarkable similarity between translation termination and ribosome rescue mechanisms. This review describes how these pathways rely on inherent ribosome dynamics, emphasizing the active role of the ribosome in all translation steps.
Subject(s)
Bacteria/metabolism , Cytoplasm/metabolism , Mitochondria/metabolism , Ribosomes/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carboxylic Ester Hydrolases/metabolism , Peptide Chain Termination, Translational , Protein Biosynthesis , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolismABSTRACT
Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-Gâ¢GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.
Subject(s)
Frameshifting, Ribosomal/genetics , Peptide Chain Elongation, Translational/genetics , Peptide Elongation Factor G/genetics , RNA, Messenger/genetics , RNA, Transfer/genetics , Ribosomes/genetics , Biocatalysis , Cryoelectron Microscopy , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Models, Molecular , Nucleic Acid Conformation , Peptide Elongation Factor G/chemistry , Peptide Elongation Factor G/metabolism , Protein Conformation , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/metabolism , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Ribosomes/metabolism , Ribosomes/ultrastructure , tRNA Methyltransferases/genetics , tRNA Methyltransferases/metabolismABSTRACT
Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ribosomes stalled on mRNAs truncated at the A site, allowing ribosome recycling. Prior structural work revealed that ArfB recognizes such ribosomes by inserting its C-terminal α-helix into the vacant mRNA tunnel. In this work, we report that ArfB can efficiently recognize a wider range of mRNA substrates, including longer mRNAs that extend beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on the mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 Å and are stabilized in the intersubunit space by dimeric ArfB. Uncovering distinct modes of ArfB function resolves conflicting biochemical and structural studies, and may lead to re-examination of other ribosome rescue pathways, whose functions depend on mRNA lengths.
Subject(s)
Escherichia coli Proteins/metabolism , RNA, Messenger/metabolism , Ribosomes/metabolism , Biocatalysis , Dimerization , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/ultrastructure , Models, Biological , Protein Conformation , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/ultrastructure , Ribosome Subunits/metabolism , Ribosomes/ultrastructureABSTRACT
Termination of protein biosynthesis is an essential step of gene expression, during which a complete functional protein is released from the ribosome. Premature or inefficient termination results in truncated, nonfunctional, or toxic proteins that may cause disease. Indeed, more than 10% of human genetic diseases are caused by nonsense mutations leading to premature termination. Efficient and sensitive approaches are required to study eukaryotic termination mechanisms and to identify potential therapeutics that modulate termination. Canonical radioactivity-based termination assays are complex, report on a short peptide release, and are incompatible with high-throughput screening. Here we describe a robust and simple in vitro assay to study the kinetics of full-protein release. The assay monitors luminescence upon release of nanoluciferase from a mammalian pretermination complex. The assay can be used to record time-progress curves of protein release in a high-throughput format, making it optimal for studying release kinetics and for high-throughput screening for small molecules that modulate the efficiency of termination.
Subject(s)
Biological Assay/methods , Luciferases/metabolism , Peptide Termination Factors/metabolism , Protein Biosynthesis , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Humans , Peptide Chain Termination, TranslationalABSTRACT
Ribosomes accurately decode mRNA by proofreading each aminoacyl-tRNA that is delivered by the elongation factor EF-Tu1. To understand the molecular mechanism of this proofreading step it is necessary to visualize GTP-catalysed elongation, which has remained a challenge2-4. Here we use time-resolved cryogenic electron microscopy to reveal 33 ribosomal states after the delivery of aminoacyl-tRNA by EF-Tuâ¢GTP. Instead of locking cognate tRNA upon initial recognition, the ribosomal decoding centre dynamically monitors codon-anticodon interactions before and after GTP hydrolysis. GTP hydrolysis enables the GTPase domain of EF-Tu to extend away, releasing EF-Tu from tRNA. The 30S subunit then locks cognate tRNA in the decoding centre and rotates, enabling the tRNA to bypass 50S protrusions during accommodation into the peptidyl transferase centre. By contrast, the decoding centre fails to lock near-cognate tRNA, enabling the dissociation of near-cognate tRNA both during initial selection (before GTP hydrolysis) and proofreading (after GTP hydrolysis). These findings reveal structural similarity between ribosomes in initial selection states5,6 and in proofreading states, which together govern the efficient rejection of incorrect tRNA.
Subject(s)
Cryoelectron Microscopy , Guanosine Triphosphate/metabolism , Peptide Elongation Factor Tu/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Ribosomes/metabolism , Ribosomes/ultrastructure , Escherichia coli , GTP Phosphohydrolases/metabolism , Guanosine Diphosphate/chemistry , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/chemistry , Hydrolysis , Models, Molecular , Peptide Elongation Factor Tu/chemistry , Peptide Elongation Factor Tu/ultrastructure , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer/chemistry , RNA, Transfer/ultrastructure , Ribosomes/chemistry , RotationABSTRACT
Recombinant adeno-associated viruses (rAAVs) are currently considered the safest and most reliable gene delivery vehicles for human gene therapy. Three serotype capsids, AAV1, AAV2, and AAV9, have been approved for commercial use in patients, but they may not be suitable for all therapeutic contexts. Here, we describe a novel capsid identified in a human clinical sample by high-throughput, long-read sequencing. The capsid, which we have named AAVv66, shares high sequence similarity with AAV2. We demonstrate that compared to AAV2, AAVv66 exhibits enhanced production yields, virion stability, and CNS transduction. Unique structural properties of AAVv66 visualized by cryo-EM at 2.5-Å resolution, suggest that critical residues at the three-fold protrusion and at the interface of the five-fold axis of symmetry likely contribute to the beneficial characteristics of AAVv66. Our findings underscore the potential of AAVv66 as a gene therapy vector.
Subject(s)
Capsid Proteins/genetics , Capsid/metabolism , Dependovirus/genetics , Genetic Vectors/genetics , Animals , Capsid/ultrastructure , Capsid Proteins/classification , Central Nervous System/virology , Cryoelectron Microscopy , DNA, Viral/analysis , DNA, Viral/genetics , Dependovirus/classification , Dependovirus/physiology , High-Throughput Nucleotide Sequencing/methods , Humans , Mice, Inbred C57BL , Mice, Transgenic , Phylogeny , Serogroup , Transduction, Genetic , Virus Assembly/geneticsABSTRACT
5S rRNA is an indispensable component of cytoplasmic ribosomes in all species. The functions of 5S rRNA and the reasons for its evolutionary preservation as an independent molecule remain unclear. Here we used ribosome engineering to investigate whether 5S rRNA autonomy is critical for ribosome function and cell survival. By linking circularly permutated 5S rRNA with 23S rRNA we generated a bacterial strain devoid of free 5S rRNA. Viability of the engineered cells demonstrates that autonomous 5S rRNA is dispensable for cell growth under standard conditions and is unlikely to have essential functions outside the ribosome. The fully assembled ribosomes carrying 23S-5S rRNA are highly active in translation. However, the engineered cells accumulate aberrant 50S subunits unable to form stable 70S ribosomes. Cryo-EM analysis revealed a malformed peptidyl transferase center in the misassembled 50S subunits. Our results argue that the autonomy of 5S rRNA is preserved due to its role in ribosome biogenesis.
Subject(s)
RNA, Ribosomal, 5S/metabolism , Ribosomes/metabolism , Catalytic Domain , Cryoelectron Microscopy , DNA-Binding Proteins/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Gene Expression Regulation , Genetic Engineering , Mutation , Nucleic Acid Conformation , Peptidyl Transferases/metabolism , RNA, Bacterial , RNA, Ribosomal, 23S/metabolism , Rec A Recombinases/metabolism , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Bacterial/metabolismABSTRACT
Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.
Subject(s)
Nucleic Acid Conformation , RNA, Messenger/metabolism , RNA, Transfer/metabolism , Ribosomes , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cryoelectron Microscopy , DNA Polymerase III/genetics , DNA Polymerase III/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Frameshifting, Ribosomal , Fusion Proteins, gag-pol , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Viral , HIV-1/genetics , HIV-1/metabolism , RNA, Bacterial , RNA, Messenger/chemistry , RNA, Transfer/chemistryABSTRACT
Protein synthesis ends when a ribosome reaches an mRNA stop codon. Release factors (RFs) decode the stop codon, hydrolyze peptidyl-tRNA to release the nascent protein, and then dissociate to allow ribosome recycling. To visualize termination by RF2, we resolved a cryo-EM ensemble of E. coli 70Sâ¢RF2 structures at up to 3.3 Å in a single sample. Five structures suggest a highly dynamic termination pathway. Upon peptidyl-tRNA hydrolysis, the CCA end of deacyl-tRNA departs from the peptidyl transferase center. The catalytic GGQ loop of RF2 is rearranged into a long ß-hairpin that plugs the peptide tunnel, biasing a nascent protein toward the ribosome exit. Ribosomal intersubunit rotation destabilizes the catalytic RF2 domain on the 50S subunit and disassembles the central intersubunit bridge B2a, resulting in RF2 departure. Our structures visualize how local rearrangements and spontaneous inter-subunit rotation poise the newly-made protein and RF2 to dissociate in preparation for ribosome recycling.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Peptide Chain Termination, Translational , Peptide Termination Factors/metabolism , Ribosomes/metabolism , Cryoelectron Microscopy , Escherichia coli Proteins/chemistry , Peptide Termination Factors/chemistry , Ribosomes/chemistryABSTRACT
Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.
Subject(s)
Anemia, Diamond-Blackfan/genetics , Adolescent , Child , Child, Preschool , Cohort Studies , Exome/genetics , Exons/genetics , Female , Gene Deletion , Genetic Association Studies/methods , Humans , Intercellular Signaling Peptides and Proteins/genetics , Male , Mutation/genetics , Phenotype , Ribosomal Proteins/genetics , Ribosomes/genetics , Sequence Analysis, RNA/methods , Exome Sequencing/methodsABSTRACT
Accurate translation termination by release factors (RFs) is critical for the integrity of cellular proteomes. Premature termination on sense codons, for example, results in truncated proteins, whose accumulation could be detrimental to the cell. Nevertheless, some sense codons are prone to triggering premature termination, but the structural basis for this is unclear. To investigate premature termination, we determined a cryo-EM structure of the Escherichia coli 70S ribosome bound with RF1 in response to a UAU (Tyr) sense codon. The structure reveals that RF1 recognizes a UAU codon similarly to a UAG stop codon, suggesting that sense codons induce premature termination because they structurally mimic a stop codon. Hydrophobic interaction between the nucleobase of U3 (the third position of the UAU codon) and conserved Ile-196 in RF1 is important for misreading the UAU codon. Analyses of RNA binding in ribonucleoprotein complexes or by amino acids reveal that Ile-U packing is a frequent protein-RNA-binding motif with key functional implications. We discuss parallels with eukaryotic translation termination by the release factor eRF1.