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.
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson’s disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light ...and electron microscopy, in situ cryo-electron tomography, and subtomogram analysis, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase and kinase are in close proximity, with the GTPase closer to the microtubule surface, whereas the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to generate models of previously unsolved structures in their cellular environment.
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•Cryo-electron tomography and integrative modeling reveal LRRK2’s structure in situ•Parkinson’s disease-linked LRRK2 forms double-helical filaments around microtubules•Homotypic interactions between the WD40 and COR domains form the filaments•The GTPase and kinase domains face the microtubule and cytoplasm, respectively
Cryo-electron tomography combined with integrative modeling yields the structure of LRKK2 in a cellular context and shows how filaments of the protein embrace microtubules.
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.
Synopsis
A combination of in vitro biochemistry, ribosome profiling, and cryo‐EM defines molecular events that lead to ribosome stalling at inhibitory codon combinations and poly(A) tracts, demonstrating novel mRNA‐induced mechanisms slowing translational elongation by eukaryotic ribosomes.
Inhibitory codon pairs and poly(A) tracts within mRNAs form decoding‐incompatible conformations in the decoding center of the ribosome.
Direct effects of these structures on decoding are documented by high‐resolution ribosome profiling, kinetic studies and detailed cryo‐EM structural analysis.
These data demonstrate a novel mRNA‐induced mechanism of translational stalling in eukaryotic ribosomes.
The cryo‐EM structure of poly(A)‐stalled disomes reveals a novel conformation for collided ribosomes that rationalizes translational frameshifting.
A combination of in vitro biochemistry, ribosome profiling, and cryo‐EM reveals that elongation‐slowing mRNA elements stall ribosomes in decoding‐incompatible RNA conformations in the ribosomal decoding center.
Ribosome-associated quality control (RQC) is a conserved process degrading potentially toxic truncated nascent peptides whose malfunction underlies neurodegeneration and proteostasis decline in ...aging. During RQC, dissociation of stalled ribosomes is followed by elongation of the nascent peptide with alanine and threonine residues, driven by Rqc2 independently of mRNA, the small ribosomal subunit and guanosine triphosphate (GTP)-hydrolyzing factors. The resulting CAT tails (carboxy-terminal tails) and ubiquitination by Ltn1 mark nascent peptides for proteasomal degradation. Here we present ten cryogenic electron microscopy (cryo-EM) structures, revealing the mechanistic basis of individual steps of the CAT tailing cycle covering initiation, decoding, peptidyl transfer, and tRNA translocation. We discovered eIF5A as a crucial eukaryotic RQC factor enabling peptidyl transfer. Moreover, we observed dynamic behavior of RQC factors and tRNAs allowing for processivity of the CAT tailing cycle without additional energy input. Together, these results elucidate key differences as well as common principles between CAT tailing and canonical translation.
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•Ten cryo-EM structures reveal the molecular mechanism of the CAT tailing cycle•EIF5A, a universal translation-promoting factor, is a eukaryotic RQC factor•Two cryo-EM structures show the dynamic mode of interaction between Ltn1 and 60S
Tesina et al. use cryo-EM to reveal the structural mechanism of CAT tailing, a key process in degradation of aberrant nascent proteins taking place at the large ribosomal subunit. They reveal common principles and important differences between CAT tailing and canonical translation from initiation, decoding, and peptidyl transfer to termination.
Abstract Tigecycline is widely used for treating complicated bacterial infections for which there are no effective drugs. It inhibits bacterial protein translation by blocking the ribosomal A-site. ...However, even though it is also cytotoxic for human cells, the molecular mechanism of its inhibition remains unclear. Here, we present cryo-EM structures of tigecycline-bound human mitochondrial 55S, 39S, cytoplasmic 80S and yeast cytoplasmic 80S ribosomes. We find that at clinically relevant concentrations, tigecycline effectively targets human 55S mitoribosomes, potentially, by hindering A-site tRNA accommodation and by blocking the peptidyl transfer center. In contrast, tigecycline does not bind to human 80S ribosomes under physiological concentrations. However, at high tigecycline concentrations, in addition to blocking the A-site, both human and yeast 80S ribosomes bind tigecycline at another conserved binding site restricting the movement of the L1 stalk. In conclusion, the observed distinct binding properties of tigecycline may guide new pathways for drug design and therapy.
Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation
. In bacteria, it remains unclear how rescue pathways distinguish ribosomes ...stalled in the middle of a transcript from actively translating ribosomes
. 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 mRNAs
) 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.
Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation ...followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal the mechanism of its mRNA-dependent and nascent peptide-dependent translational stalling. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent polyubiquitination of collided disomes and, preferentially, trisomes. The distinct trisome architecture, visualized using cryo-EM, provides the structural basis for the more-efficient recognition by Hel2 compared with that of disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled polyubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.
We observed the assembly of a nucleus-like structure in bacteria during viral infection. Using fluorescence microscopy and cryo-electron tomography, we showed that Pseudomonas chlororaphis phage ...201φ2-1 assembled a compartment that separated viral DNA from the cytoplasm. The phage compartment was centered by a bipolar tubulin-based spindle, and it segregated phage and bacterial proteins according to function. Proteins involved in DNA replication and transcription localized inside the compartment, whereas proteins involved in translation and nucleotide synthesis localized outside. Later during infection, viral capsids assembled on the cytoplasmic membrane and moved to the surface of the compartment for DNA packaging. Ultimately, viral particles were released from the compartment and the cell lysed. These results demonstrate that phages have evolved a specialized structure to compartmentalize viral replication.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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