70S Ribosome Research Articles

The context of the ribosome binding site in mRNAs defines specificity of action of kasugamycin, an inhibitor of translation initiation

Kasugamycin (KSG) is an aminoglycoside antibiotic widely used in agriculture and exhibits considerable medical potential. Previous studies suggested that KSG interferes with translation by blocking binding of canonical messenger RNA (mRNA) and initiator transfer tRNA (tRNA) to the small ribosomal subunit, thereby preventing initiation of protein synthesis. Here, by using genome-wide approaches, we show that KSG can interfere with translation even after the formation of the 70S initiation complex on mRNA, as the extent of KSG-mediated translation inhibition correlates with increased occupancy of start codons by 70S ribosomes. Even at saturating concentrations, KSG does not completely abolish translation, allowing for continuing expression of some Escherichia coli proteins. Differential action of KSG significantly depends on the nature of the mRNA residue immediately preceding the start codon, with guanine in this position being the most conducive to inhibition by the drug. In addition, the activity of KSG is attenuated by translational coupling as genes whose start codons overlap with the coding regions or the stop codons of the upstream cistrons tend to be less susceptible to drug-mediated inhibition. Altogether, our findings reveal KSG as an example of a small ribosomal subunit-targeting antibiotic with a well-pronounced context specificity of action.

Interplay between an ATP-binding cassette F protein and the ribosome from Mycobacterium tuberculosis

EttA, energy-dependent translational throttle A, is a ribosomal factor that gates ribosome entry into the translation elongation cycle. A detailed understanding of its mechanism of action is limited due to the lack of high-resolution structures along its ATPase cycle. Here we present the cryo-electron microscopy (cryo-EM) structures of EttA from Mycobacterium tuberculosis (Mtb), referred to as MtbEttA, in complex with the Mtb 70S ribosome initiation complex (70SIC) at the pre-hydrolysis (ADPNP) and transition (ADP-VO4) states, and the crystal structure of MtbEttA alone in the post-hydrolysis (ADP) state. We observe that MtbEttA binds the E-site of the Mtb 70SIC, remodeling the P-site tRNA and the ribosomal intersubunit bridge B7a during the ribosomal ratcheting. In return, the rotation of the 30S causes conformational changes in MtbEttA, forcing the two nucleotide-binding sites (NBSs) to alternate to engage each ADPNP in the pre-hydrolysis states, followed by complete engagements of both ADP-VO4 molecules in the ATP-hydrolysis transition states. In the post-hydrolysis state, the conserved ATP-hydrolysis motifs of MtbEttA dissociate from both ADP molecules, leaving two nucleotide-binding domains (NBDs) in an open conformation. These structures reveal a dynamic interplay between MtbEttA and the Mtb ribosome, providing insights into the mechanism of translational regulation by EttA-like proteins.

The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase binding in 40S ribosome assembly.

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AFs) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate "handover" from one highly related AF to another, remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the binding of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1 into its functional site. Inactive Tsr3 blocks Rio1 function, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

A novel ribosome-dimerization protein found in the hyperthermophilic archaeon Pyrococcus furiosus using ribosome-associated proteomics

Ribosome dimerization is one of the bacterial events that suppresses protein synthesis in the stationary phase. Protein factors responsible for ribosome dimerization in bacteria are well characterized, whereas no information is available for the corresponding factors in archaeal and eukaryotic cells. Here we describe a protein found among the ribosome-associated proteins which dimerizes the 30S ribosomal subunit of the archaeon Pyrococcus furiosus. The ribosome-associated proteins were prepared by high-salt wash of crude ribosomes, and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Of the detected proteins we focused on a protein (PF0560) whose Protein Score was the highest of all of the function-unknown proteins. PF0560 protein had a pronounced effect on the sedimentation pattern of the 30S ribosomal subunit; addition of this protein to isolated 30S subunit reduced the 30S fraction and increased the amount of the 50S fraction. This increase presumably corresponds to the dimer of the 30S subunit. The PF0560-dependent 30S-dimerization, was also observed by gel electrophoretic analysis. This effect was not observed in EDTA-treated 30S subunit, with protein-free 16S rRNA or with bacterial/eukaryotic ribosomal small subunits. Furthermore, PF0560 protein suppressed the formation of functional 70S ribosomes. These results suggest that PF0560 is a novel 30S dimerization factor, which might participate in regulation of archaeal translation.

Effect of Amino Acid Substitutions on 70S Ribosomal Binding, Cellular Uptake, and Antimicrobial Activity of Oncocin Onc112.

Proline‐rich antimicrobial peptides (PrAMPs) are promising candidates for the treatment of infections caused by high‐priority human pathogens. Their mode of action consists of (I) passive diffusion across the outer membrane, (II) active transport through the inner membrane, and (III) inhibition of protein biosynthesis by blocking the exit tunnel of the 70S ribosome. We tested whether in vitro data on ribosomal binding and bacterial uptake could predict the antibacterial activity of PrAMPs against Gram‐negative and Gram‐positive bacteria. Ribosomal binding and bacterial uptake rates were measured for 47 derivatives of PrAMP Onc112 and compared to the minimal inhibitory concentrations (MIC) of each peptide. Ribosomal binding was evaluated for ribosome extracts from four Gram‐negative bacteria. Bacterial uptake was assessed by quantifying each peptide in the supernatants of bacterial cultures. Oncocin analogues with a higher net positive charge appeared to be more active, although their ribosome binding and uptake rates were not necessarily better than for Onc112. The data suggest a complex mode of action influenced by further factors improving or reducing the antibacterial activity, including diffusion through membranes, transport mechanism, secondary targets, off‐target binding, intracellular distribution, and membrane effects. Relying only on in vitro binding and uptake data may not be sufficient for the rational development of more active analogues.

Puf-A promotes cancer progression by interacting with nucleophosmin in nucleolus

Previously, we identified Puf-A as a novel member of Puf-family RNA-binding proteins; however, its biological functions remain obscure. Analysis of tumor samples of non-small cell lung cancer (NSCLC) showed that high Puf-A expression correlated with high histology grade and abnormal p53 status. Kaplan–Meier curve for overall survival revealed high expression of Puf-A to predict poor prognosis in stage I NSCLC. Among patients with colorectal cancer, high Puf-A expression also showed an adverse impact on overall survival. In lung cancer cell lines, downregulation of p53 increased Puf-A expression, and upregulation of p53 dampened its expression. However, luciferase reporter assays indicated that PUF-A locus harbored the p53-response element, but regulated Puf-A transcription indirectly. In vivo suppression of p53 in CCSP-rtTA/TetO-Cre/LSL-KrasG12D/p53flox/flox conditional mutant mice accelerated the progression of the KrasG12D-driven lung cancer, along with enhanced expression of Puf-A. Importantly, intranasal delivery of shPuf-A to the inducible KrasG12D/p53flox/flox mice suppressed tumor progression. Puf-A silencing led to marked decreases in the 80S ribosomes, along with decrease in S6 and L5 in the cytoplasm and accumulation in the nucleolus. Based on immunofluorescence staining and immunoprecipitation studies, Puf-A interacted with NPM1 in nucleolus. Puf-A silencing resulted in NPM1 translocation from nucleolus to nucleoplasm and this disruption of NPM1 localization was reversed by a rescue experiment. Mechanistically, Puf-A silencing altered NPM1 localization, leading to the retention of ribosomal proteins in nucleolus and diminished ribosome biogenesis, followed by cell-cycle arrest/cell death. Puf-A is a potential theranostic target for cancer therapy and an important player in cancer progression.

Ribosomal leaky scanning through a translated uORF requires eIF4G2.

eIF4G2 (DAP5 or Nat1) is a homologue of the canonical translation initiation factor eIF4G1 in higher eukaryotes but its function remains poorly understood. Unlike eIF4G1, eIF4G2 does not interact with the cap-binding protein eIF4E and is believed to drive translation under stress when eIF4E activity is impaired. Here, we show that eIF4G2 operates under normal conditions as well and promotes scanning downstream of the eIF4G1-mediated 40S recruitment and cap-proximal scanning. Specifically, eIF4G2 facilitates leaky scanning for a subset of mRNAs. Apparently, eIF4G2 replaces eIF4G1 during scanning of 5′ UTR and the necessity for eIF4G2 only arises when eIF4G1 dissociates from the scanning complex. In particular, this event can occur when the leaky scanning complexes interfere with initiating or elongating 80S ribosomes within a translated uORF. This mechanism is therefore crucial for higher eukaryotes which are known to have long 5′ UTRs with highly frequent uORFs. We suggest that uORFs are not the only obstacle on the way of scanning complexes towards the main start codon, because certain eIF4G2 mRNA targets lack uORF(s). Thus, higher eukaryotes possess two distinct scanning complexes: the principal one that binds mRNA and initiates scanning, and the accessory one that rescues scanning when the former fails.

Quantitative mining of compositional heterogeneity in cryo-EM datasets of ribosome assembly intermediates

Single-particle cryoelectron microscopy (cryo-EM) offers a unique opportunity to characterize macromolecular structural heterogeneity by virtue of its ability to place distinct particle populations into different groups through computational classification. However, there is a dearth of tools for surveying the heterogeneity landscape, quantitatively analyzing heterogeneous particle populations after classification, deciding how many unique classes are represented by the data, and accurately cross-comparing reconstructions. Here, we develop a workflow that contains discovery and analysis modules to quantitatively mine cryo-EM data for sets of structures with maximal diversity. This workflow was applied to a dataset of E.coli 50S ribosome assembly intermediates, which are characterized by significant structural heterogeneity. We identified more detailed branchpoints in the assembly process and characterized the interactions of an assembly factor with immature intermediates. While the tools described here were developed for ribosome assembly, they should be broadly applicable to the analysis of other heterogeneous cryo-EM datasets.

Urdnet: A Cryo-EM Particle Automatic Picking Method

Cryo-Electron Microscopy (Cryo-EM) images are characterized by the low signal-to-noise ratio, low contrast, serious background noise, more impurities, less data, difficult data labeling, simpler image semantics, and relatively fixed structure, while U-Net obtains low resolution when downsampling rate information to complete object category recognition, obtains high-resolution information during upsampling to complete precise segmentation and positioning, fills in the underlying information through skip connection to improve the accuracy of image segmentation, and has advantages in biological image processing like Cryo-EM image. This article proposes A U-Net based residual intensive neural network (Urdnet), which combines point-level and pixel-level tags, used to accurately and automatically locate particles from cryo-electron microscopy images, and solve the bottleneck that cryo-EM Single-particle biological macromolecule reconstruction requires tens of thousands of automatically picked particles. The 80S ribosome, HCN1 channel and TcdA1 toxin subunits, and other public protein datasets have been trained and tested on Urdnet. The experimental results show that Urdnet could reach the same excellent particle picking performances as the mainstream methods of RELION, DeepPicker, and acquire the 3D structure of picked particles with higher resolution.

Recovery of Conformational Continuum From Single-Particle Cryo-EM Images: Optimization of ManifoldEM Informed by Ground Truth.

This work is based on the manifold-embedding approach to study biological molecules exhibiting continuous conformational changes. Previous work established a method—now termed ManifoldEM—capable of reconstructing 3D movies and accompanying free-energy landscapes from single-particle cryo-EM images of macromolecules exercising multiple conformational degrees of freedom. While ManifoldEM has proven its viability in several experimental studies, critical limitations and uncertainties have been found throughout its extended development and use. Guided by insights from studies with cryo-EM ground-truth data, simulated from atomic structures undergoing conformational changes, we have built a novel framework, ESPER, able to retrieve the free-energy landscape and respective 3D Coulomb potential maps for all states simulated. As shown by a direct comparison of ground truth vs. recovered maps, and analysis of experimental data from the 80S ribosome and ryanodine receptor, ESPER offers substantial improvements relative to the previous work.

Ribosomal Hibernation-Associated Factors in Escherichia coli.

Bacteria convert active 70S ribosomes to inactive 100S ribosomes to survive under various stress conditions. This state, in which the ribosome loses its translational activity, is known as ribosomal hibernation. In gammaproteobacteria such as Escherichia coli, ribosome modulation factor and hibernation-promoting factor are involved in forming 100S ribosomes. The expression of ribosome modulation factor is regulated by (p)ppGpp (which is induced by amino acid starvation), cAMP-CRP (which is stimulated by reduced metabolic energy), and transcription factors involved in biofilm formation. This indicates that the formation of 100S ribosomes is an important strategy for bacterial survival under various stress conditions. In recent years, the structures of 100S ribosomes from various bacteria have been reported, enhancing our understanding of the 100S ribosome. Here, we present previous findings on the 100S ribosome and related proteins and describe the stress-response pathways involved in ribosomal hibernation.

Deficiency of the Ribosomal Protein uL5 Leads to Significant Rearrangements of the Transcriptional and Translational Landscapes in Mammalian Cells.

Protein uL5 (formerly called L11) is an integral component of the large (60S) subunit of the human ribosome, and its deficiency in cells leads to the impaired biogenesis of 60S subunits. Using RNA interference, we reduced the level of uL5 in HEK293T cells by three times, which caused an almost proportional decrease in the content of the fraction corresponding to 80S ribosomes, without a noticeable diminution in the level of polysomes. By RNA sequencing of uL5-deficient and control cell samples, which were those of total mRNA and mRNA from the polysome fraction, we identified hundreds of differentially expressed genes (DEGs) at the transcriptome and translatome levels and revealed dozens of genes with altered translational efficiency (GATEs). Transcriptionally up-regulated DEGs were mainly associated with rRNA processing, pre-mRNA splicing, translation and DNA repair, while down-regulated DEGs were genes of membrane proteins; the type of regulation depended on the GC content in the 3′ untranslated regions of DEG mRNAs. The belonging of GATEs to up-regulated and down-regulated ones was determined by the coding sequence length of their mRNAs. Our findings suggest that the effects observed in uL5-deficient cells result from an insufficiency of translationally active ribosomes caused by a deficiency of 60S subunits.

Probing Interplays between Human XBP1u Translational Arrest Peptide and 80S Ribosome.

The ribosome stalling mechanism is a crucial biological process, yet its atomistic underpinning is still elusive. In this framework, the human XBP1u translational arrest peptide (AP) plays a central role in regulating the unfolded protein response (UPR) in eukaryotic cells. Here, we report multimicrosecond all-atom molecular dynamics simulations designed to probe the interactions between the XBP1u AP and the mammalian ribosome exit tunnel, both for the wild type AP and for four mutant variants of different arrest potencies. Enhanced sampling simulations allow investigating the AP release process of the different variants, shedding light on this complex mechanism. The present outcomes are in qualitative/quantitative agreement with available experimental data. In conclusion, we provide an unprecedented atomistic picture of this biological process and clear-cut insights into the key AP–ribosome interactions.

Accuracy mechanism of eukaryotic ribosome translocation

Translation of the genetic code into proteins is realized through repetitions of synchronous translocation of messenger RNA (mRNA) and transfer RNAs (tRNA) through the ribosome. In eukaryotes translocation is ensured by elongation factor 2 (eEF2), which catalyses the process and actively contributes to its accuracy1. Although numerous studies point to critical roles for both the conserved eukaryotic posttranslational modification diphthamide in eEF2 and tRNA modifications in supporting the accuracy of translocation, detailed molecular mechanisms describing their specific functions are poorly understood. Here we report a high-resolution X-ray structure of the eukaryotic 80S ribosome in a translocation-intermediate state containing mRNA, naturally modified eEF2 and tRNAs. The crystal structure reveals a network of stabilization of codon–anticodon interactions involving diphthamide1 and the hypermodified nucleoside wybutosine at position 37 of phenylalanine tRNA, which is also known to enhance translation accuracy2. The model demonstrates how the decoding centre releases a codon–anticodon duplex, allowing its movement on the ribosome, and emphasizes the function of eEF2 as a ‘pawl’ defining the directionality of translocation3. This model suggests how eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs undergo large-scale molecular reorganizations to ensure maintenance of the mRNA reading frame during the complex process of translocation.

Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch

Programmed –1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.

Molecular Basis of SARS-CoV-2 Nsp1-Induced Immune Translational Shutdown as Revealed by All-Atom Simulations.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic represents the most severe global health crisis in modern human history. One of the major SARS-CoV-2 virulence factors is nonstructural protein 1 (Nsp1), which, outcompeting with the binding of host mRNA to the human ribosome, triggers a translation shutdown of the host immune system. Here, microsecond-long all-atom simulations of the C-terminal portion of the SARS-CoV-2/SARS-CoV Nsp1 in complex with the 40S ribosome disclose that SARS-CoV-2 Nsp1 has evolved from its SARS-CoV ortholog to more effectively hijack the ribosome by undergoing a critical switch of Q/E158 and E/Q159 residues that perfects Nsp1's interactions with the ribosome. Our outcomes offer a basis for understanding the sophisticated mechanisms underlying SARS-CoV-2 diversion and exploitation of human cell components to its deadly purposes.

Functionally distinct roles for eEF2K in the control of ribosome availability and p-body abundance

Processing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splitting in vitro. Collectively, the data suggest that eEF2K defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons.

Nsp1 of SARS-CoV-2 stimulates host translation termination

ABSTRACT Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.

The Stringent Response Inhibits 70S Ribosome Formation in Staphylococcus aureus by Impeding GTPase-Ribosome Interactions.

ABSTRACTDuring nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of a stress signaling network termed the stringent response. RsgA, RbgA, Era, and HflX are four ribosome-associated GTPases (RA-GTPases) that bind to (p)ppGpp in Staphylococcus aureus. These enzymes are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) ribosome-associated states. Entry into the OFF state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly and growth. Here, we determine how (p)ppGpp impacts RA-GTPase-ribosome interactions. We show that RA-GTPases preferentially bind to 5′-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell to shut down ribosome biogenesis during stress. Stopped-flow fluorescence and association assays reveal that when bound to (p)ppGpp, the association of RA-GTPases to ribosomal subunits is destabilized, both in vitro and within bacterial cells. Consistently, structural analysis of the ppGpp-bound RA-GTPase RsgA reveals an OFF-state conformation similar to the GDP-bound state, with the G2/switch I loop adopting a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated ribosome assembly and growth control.

In vitro reconstitution of the Escherichia coli 70S ribosome with a full set of recombinant ribosomal proteins.

Many studies of the reconstitution of the Escherichia coli small ribosomal subunit from its individual molecular parts have been reported, but contrastingly, similar studies of the large ribosomal subunit have not been well performed to date. Here, we describe protocols for preparing the 33 ribosomal proteins of the E. coli 50S subunit and demonstrate successful reconstitution of a functionally active 50S particle that can perform protein synthesis in vitro. We also successfully reconstituted both ribosomal subunits (30S and 50S) and 70S ribosomes using a full set of recombinant ribosomal proteins by integrating our developed method with the previously developed fully recombinant-based integrated synthesis, assembly and translation. The approach described here makes a major contribution to the field of ribosome engineering and could be fundamental to the future studies of ribosome assembly processes.