Ribosomal Tunnel Research Articles

Shifting frames to make more proteins

Coronavirus Severe acute respiratory syndrome coronavirus 2 critically depends on the ribosomal frameshifting that occurs between two large open reading frames in its genomic RNA for expression of viral replicase. Programmed frameshifting occurs during translation, when the ribosome encounters a stimulatory pseudoknot RNA fold. Using a combination of cryo–electron microscopy and biochemistry, Bhatt et al. revealed that the pseudoknot resists unfolding as it lodges at the entry of the ribosomal messenger RNA channel. This causes back slippage of the viral RNA, resulting in a minus-1 shift of the reading frame of translation. A partially folded nascent viral polyprotein forms specific interactions inside the ribosomal tunnel that can influence the efficiency of frameshifting. Science , abf3546, this issue p. [1306][1] [1]: /lookup/doi/10.1126/science.abf3546

Fluorescence Anisotropy Decays and Microscale-Volume Viscometry Reveal the Compaction of Ribosome-Bound Nascent Proteins.

This work introduces a technology that combines fluorescence anisotropy decay with microscale-volume viscometry to investigate the compaction and dynamics of ribosome-bound nascent proteins. Protein folding in the cell, especially when nascent chains emerge from the ribosomal tunnel, is poorly understood. Previous investigations based on fluorescence anisotropy decay determined that a portion of the ribosome-bound nascent protein apomyoglobin (apoMb) forms a compact structure. This work, however, could not assess the size of the compact region. The combination of fluorescence anisotropy with microscale-volume viscometry, presented here, enables identifying the size of compact nascent-chain subdomains using a single fluorophore label. Our results demonstrate that the compact region of nascent apoMb contains 57-83 amino acids and lacks residues corresponding to the two native C-terminal helices. These amino acids are necessary for fully burying the nonpolar residues in the native structure, yet they are not available for folding before ribosome release. Therefore, apoMb requires a significant degree of post-translational folding for the generation of its native structure. In summary, the combination of fluorescence anisotropy decay and microscale-volume viscometry is a powerful approach to determine the size of independently tumbling compact regions of biomolecules. This technology is of general applicability to compact macromolecules linked to larger frameworks.

Structural polymorphism and substrate promiscuity of a ribosome-associated molecular chaperone.

Trigger factor (TF) is a highly conserved multi-domain molecular chaperone that exerts its chaperone activity at the ribosomal tunnel exit from which newly synthesized nascent chains emerge. TF also displays promiscuous substrate binding for a large number of cytosolic proteins independent of ribosome binding. We asked how TF recognizes a variety of substrates while existing in a monomer-dimer equilibrium. Paramagnetic nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy were used to show that dimeric TF displays a high degree of structural polymorphism in solution. A series of peptides has been generated to quantify their TF binding affinities in relation with their sequence compositions. The results confirmed a previous predication that TF preferentially binds to peptide fragments that are rich in aromatic and positively charged amino acids. NMR paramagnetic relaxation enhancement analysis showed that TF utilizes multiple binding sites, located in the chaperone domain and part of the prolyl trans-cis isomerization domain, to interact with these peptides. Dimerization of TF effectively sequesters most of the substrate binding sites, which are expected to become accessible upon binding to the ribosome as a monomer. As TF lacks ATPase activity, which is commonly used to trigger conformational changes within molecular chaperones in action, the ribosome-binding-associated disassembly and conformational rearrangements may be the underlying regulatory mechanism of its chaperone activity.

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.

The ribosome modulates folding inside the ribosomal exit tunnel

Proteins commonly fold co-translationally at the ribosome, while the nascent chain emerges from the ribosomal exit tunnel. Protein domains that are sufficiently small can even fold while still located inside the tunnel. However, the effect of the tunnel on the folding dynamics of these domains is not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular dynamics simulations to investigate folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel. The tunnel is found to accelerate folding and stabilize the folded state, reminiscent of the effects of chaperonins. However, a simple mechanism involving stabilization by confinement does not explain the results. Instead, it appears that electrostatic interactions between the protein and ribosome contribute to the observed folding acceleration and stabilization of ADR1a.

Ribosome-bound Get4/5 facilitates the capture of tail-anchored proteins by Sgt2 in yeast

The guided entry of tail-anchored proteins (GET) pathway assists in the posttranslational delivery of tail-anchored proteins, containing a single C-terminal transmembrane domain, to the ER. Here we uncover how the yeast GET pathway component Get4/5 facilitates capture of tail-anchored proteins by Sgt2, which interacts with tail-anchors and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of tail-anchored proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting. Exposure of internal transmembrane domains at the tunnel exit induces high-affinity ribosome binding of SRP, which in turn prevents ribosome binding of Get4/5. In this way, the position of a transmembrane domain within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.

A Novel Approach Combining Fluorescence-Anisotropy Decays and Microviscometry to Explore the Cotranslational Compaction of Nascent Proteins

Protein folding in the cell, especially when nascent chains emerge from the ribosomal tunnel, is poorly understood. Here, we investigate the compaction and dynamics of ribosome-bound nascent single-domain proteins via a novel technology that combines fluorescence anisotropy decay with microviscometry. We targeted the model protein sperm whale apomyoglobin (ApoMb). Previous investigations based on fluorescence anisotropy decay alone determined that a portion of ribosome-bound apoMb forms a compact structure. This work, however, could not determine the size of the compact domain. In contrast, the combination of fluorescence anisotropy with microviscometry presented here enables identifying the size of compact nascent-chain subdomains. Our results demonstrate that the independently tumbling compact subdomain of nascent apoMb contains 56-81 amino acids and lacks residues corresponding to the two native C-terminal helices (denoted as G and H), which are necessary for fully burying the nonpolar residues in the native structure. Therefore, these C-terminal residues are not available for folding into the native structure until after ribosome-release. We propose that the G-helix residues may interact with the ribosome before translation termination, given their inability to sufficiently stabilize the nascent chain until the H-helix residues become available for intramolecular folding. According to a square-well-potential model envisioning local motions across a cone, the dynamics of the compact nascent-chain subdomain is spatially confined within a cone semi-angle of 15 degrees. This result highlights the highly geometrically constrained environment of the nascent protein within the ribosome. In summary, the combination of fluorescence anisotropy decay and microviscometry is a powerful approach to determine the size of independently tumbling compact regions. This technology is convenient because it requires only a single fluorophore, and can be applied to any complex biomolecule carrying an independently tumbling compact region.

The nascent polypeptide in the 60S subunit determines the Rqc2-dependency of ribosomal quality control.

Ribosome stalling at tandem CGA codons or poly(A) sequences activates quality controls for nascent polypeptides including ribosome-associated quality control (RQC) and no-go mRNA decay (NGD). In RQC pathway, Hel2-dependent uS10 ubiquitination and the RQC-trigger (RQT) complex are essential for subunit dissociation, and Ltn1-dependent ubiquitination of peptidyl-tRNA in the 60S subunit requires Rqc2. Here, we report that polytryptophan sequences induce Rqc2-independent RQC. More than 11 consecutive tryptophan residues induced RQC in a manner dependent on Hel2-mediated ribosome ubiquitination and the RQT complex. Polytryptophan sequence-mediated RQC was not coupled with CAT-tailing, and Rqc2 was not required for Ltn1-dependent degradation of the arrest products. Eight consecutive tryptophan residues located at the region proximal to the peptidyl transferase center in the ribosome tunnel inhibited CAT-tailing by tandem CGA codons. Polytryptophan sequences also induced Hel2-mediated canonical RQC-coupled NGD and RQC-uncoupled NGD outside the stalled ribosomes. We propose that poly-tryptophan sequences induce Rqc2-independent RQC, suggesting that CAT-tailing in the 60S subunit could be modulated by the polypeptide in the ribosome exit tunnel.

Water-Mediated Interactions Determine Helix Formation of Peptides in Open Nanotubes.

Water-mediated interactions (WMIs) play diverse roles in molecular biology. They are particularly relevant in geometrically confined spaces such as the interior of the chaperonin, at the interface between ligands and their binding partners, and in the ribosome tunnel. Inspired in part by the geometry of the ribosome tunnel, we consider confinement effects on the stability of peptides. We describe results from replica exchange molecular dynamics simulations of a system containing a 23-alanine or 23-serine polypeptide confined to nonpolar and polar nanotubes in the gas phase and when open to a water reservoir. We quantify the effect of water in determining the preferred conformational states of these polypeptides by calculating the difference in the solvation free energy for the helix and coil states in the open nanotube in the two phases. Our simulations reveal several possibilities. We find that nanoscopic confinement preferentially stabilizes the helical state of polypeptides with hydrophobic side chains, which is explained by the entropic stabilization mechanism proposed on the basis of polymer physics. Polypeptide chains with hydrophilic side chains can adopt helical structures within nanotubes, but helix formation is sensitive to the nature of the nanotube due to WMIs. We elaborate on the potential implications of our findings to the stability of peptides in the ribosome tunnel.

Structural and functional characterization of the N-terminal acetyltransferase Naa50

The majority of eukaryotic proteins is modified by N-terminal acetylation, which plays a fundamental role in protein homeostasis, localization, and complex formation. N-terminal acetyltransferases (NATs) mainly act co-translationally on newly synthesized proteins at the ribosomal tunnel exit. NatA is the major NAT consisting of Naa10 catalytic and Naa15 auxiliary subunits, and with Naa50 forms the NatE complex. Naa50 has recently been identified in Arabidopsis thaliana and is important for plant development and stress response regulation. Here, we determined high-resolution X-ray crystal structures of AtNaa50 in complex with AcCoA and a bisubstrate analog. We characterized its substrate specificity, determined its enzymatic parameters, and identified functionally important residues. Even though Naa50 is conserved among species, we highlight differences between Arabidopsis and yeast, where Naa50 is catalytically inactive but binds CoA conjugates. Our study provides insights into Naa50 conservation, species-specific adaptations, and serves as a basis for further studies of NATs in plants.

Exploring Allosteric Signaling in the Exit Tunnel of the Bacterial Ribosome by Molecular Dynamics Simulations and Residue Network Model.

The bacterial ribosomal tunnel is equipped with numerous sites highly sensitive to the course of the translation process. This study investigates allosteric pathways linking distant functional sites that collaboratively play a role either in translation regulation or recruitment of chaperones. We apply perturbation response scanning (PRS) analysis to 700 ns long and 500 ns long coarse-grained molecular dynamics simulations of E. coli and T. thermophilus large subunits, respectively, to reveal nucleotides/residues with the ability to transmit perturbations by dynamic rationale. We also use the residue network model with the k-shortest pathways method to calculate suboptimal pathways based on the contact topology of the ribosomal tunnel of E. coli crystal structure and 101 ClustENM generated conformers of T. thermophilus large subunit. In the upper part of the tunnel, results suggest that A2062 and A2451 can communicate in both directions for translation stalling, mostly through dynamically coupled C2063, C2064, and A2450. For a similar purpose, U2585 and U2586 are coupled with A2062, while they are also sensitive to uL4 and uL22 at the constriction region through two different pathways at the opposite sides of the tunnel wall. In addition, the constriction region communicates with the chaperone binding site on uL23 at the solvent side but through few nucleotides. Potential allosteric communication pathways between the lower part of the tunnel and chaperone binding site mostly use the flexible loop of uL23, while A1336–G1339 provide a suboptimal pathway. Both species seem to employ similar mechanisms in the long tunnel, where a non-conserved cavity at the bacterial uL23 and 23S rRNA interface is proposed as a novel drug target.

Molecular Connectivity of Mitochondrial Gene Expression and OXPHOS Biogenesis

Mitochondria contain their own gene expression systems, including membrane-bound ribosomes dedicated to synthesizing a few hydrophobic subunits of the oxidative phosphorylation (OXPHOS) complexes. We used a proximity-dependent biotinylation technique, BioID, coupled with mass spectrometry to delineate in baker's yeast a comprehensive network of factors involved in biogenesis of mitochondrial encoded proteins. This mitochondrial gene expression network (MiGENet) encompasses proteins involved in transcription, RNA processing, translation, or protein biogenesis. Our analyses indicate the spatial organization of these processes, thereby revealing basic mechanistic principles and the proteins populating strategically important sites. For example, newly synthesized proteins are directly handed over to ribosomal tunnel exit-bound factors that mediate membrane insertion, co-factor acquisition, or their mounting into OXPHOS complexes in a special early assembly hub. Collectively, the data reveal the connectivity of mitochondrial gene expression, reflecting a unique tailoring of the mitochondrial gene expression system.

Protein escape at the ribosomal exit tunnel: Effect of the tunnel shape.

We study the post-translational escape of nascent proteins at the ribosomal exit tunnel with the consideration of a real shape atomistic tunnel based on the Protein Data Bank structure of the large ribosome subunit of archeon Haloarcula marismortui. Molecular dynamics simulations employing the Go-like model for the proteins show that at intermediate and high temperatures, including a presumable physiological temperature, the protein escape process at the atomistic tunnel is quantitatively similar to that at a cylinder tunnel of length L = 72 Å and diameter d = 16 Å. At low temperatures, the atomistic tunnel, however, yields an increased probability of protein trapping inside the tunnel, while the cylinder tunnel does not cause the trapping. All-β proteins tend to escape faster than all-α proteins, but this difference is blurred on increasing the protein's chain length. A 29-residue zinc-finger domain is shown to be severely trapped inside the tunnel. Most of the single-domain proteins considered, however, can escape efficiently at the physiological temperature with the escape time distribution following the diffusion model proposed in our previous works. An extrapolation of the simulation data to a realistic value of the friction coefficient for amino acids indicates that the escape times of globular proteins are at the sub-millisecond scale. It is argued that this time scale is short enough for the smooth functioning of the ribosome by not allowing nascent proteins to jam the ribosome tunnel.

CTELS: A Cell-Free System for the Analysis of Translation Termination Rate.

Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3′ UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a “leaky” stop codon context, which likely defines the basis of nonsense suppression.

Synthesis and Antibacterial Activity of New N-Alkylammonium and Carbonate-Triazole Derivatives within Desosamine of 14- and 15-Membered Lactone Macrolides.

Desosamines of azithromycin (AZM) and clarithromycin (CLA) were modified by N-alkylation or nucleophilic substitution at the carbonyl/CuAAC sequence. Biological studies revealed a higher antibacterial potency of quaternary N-alkylammonium bromides of CLA as compared to AZM. SAR studies of CLA salts, including biological, conformation and molecular-docking analysis, enriched by physicochemical parameters, showed the importance of less bulky and unsaturated substituent for an efficient docking mode at the ribosomal tunnel and good antibacterial potency against clinical and standard Streptococcus pneumoniae and Streptococcus pyogenes strains (MICs 0.25 or 0.5 μg/mL). These CLA salts also have an at least threefold lower cytotoxicity than reference antibiotics at comparable antibacterial activity against the S. pneumoniae clinical strain. Differences in antibacterial effects noted for AZM and CLA salts bearing less bulky N-substituents can be better understood when their binding modes in the ribosomal tunnel are considered rather than their common low lipophilicity and excellent water solubility.

NAA50 Is an Enzymatically Active N α-Acetyltransferase That Is Crucial for Development and Regulation of Stress Responses.

Nα-terminal acetylation (NTA) is a prevalent protein modification in eukaryotes. In plants, the biological function of NTA remains enigmatic. The dominant N-acetyltransferase (Nat) in Arabidopsis (Arabidopsis thaliana) is NatA, which cotranslationally catalyzes acetylation of ∼40% of the proteome. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-anchoring subunit NAA15. In human (Homo sapiens), fruit fly (Drosophila melanogaster), and yeast (Saccharomyces cerevisiae), this core NatA complex interacts with NAA50 to form the NatE complex. While in metazoa, NAA50 has N-acetyltransferase activity, yeast NAA50 is catalytically inactive and positions NatA at the ribosome tunnel exit. Here, we report the identification and characterization of Arabidopsis NAA50 (AT5G11340). Consistent with its putative function as a cotranslationally acting Nat, AtNAA50-EYFP localized to the cytosol and the endoplasmic reticulum but also to the nuclei. We demonstrate that purified AtNAA50 displays Nα-terminal acetyltransferase and lysine-ε-autoacetyltransferase activity in vitro. Global N-acetylome profiling of Escherichia coli cells expressing AtNAA50 revealed conservation of NatE substrate specificity between plants and humans. Unlike the embryo-lethal phenotype caused by the absence of AtNAA10 and AtNAA15, loss of NAA50 expression resulted in severe growth retardation and infertility in two Arabidopsis transfer DNA insertion lines (naa50-1 and naa50-2). The phenotype of naa50-2 was rescued by the expression of HsNAA50 or AtNAA50. In contrast, the inactive ScNAA50 failed to complement naa50-2 Remarkably, loss of NAA50 expression did not affect NTA of known NatA substrates and caused the accumulation of proteins involved in stress responses. Overall, our results emphasize a relevant role of AtNAA50 in plant defense and development, which is independent of the essential NatA activity.

Pathway regulation mechanism revealed by cotranslational folding of villin headpiece subdomain HP35.

Cotranslational folding is one of the most important features of protein folding in vivo. Although many studies have shown that the folding pathways of cotranslational folding are different from free folding in vitro, the detailed mechanism of folding dynamics is lacking. Here we combine all-atom molecular simulations with an ideal ribosome tunnel model to investigate the cotranslational folding of villin headpiece subdomain HP35. By comparing the folding dynamics between cotranslational folding and free folding, we found that cotranslational folding tends to fold along the pathway that is easier to fold into native state in the latter. In addition, the roles of the ribosome tunnel and sequential folding are analyzed separately. Our results show that the ribosome can prevent the untimely folding of the C segment of HP35 to reduce the non-native interactions, while the translation speed can regulate the amounts of native and non-native interactions and the balance between them. Overall, these results give insights into the general mechanisms of cotranslational protein folding.

The ribosome-associated complex RAC serves in a relay that directs nascent chains to Ssb

The conserved ribosome-associated complex (RAC) consisting of Zuo1 (Hsp40) and Ssz1 (non-canonical Hsp70) acts together with the ribosome-bound Hsp70 chaperone Ssb in de novo protein folding at the ribosomal tunnel exit. Current models suggest that the function of Ssz1 is confined to the support of Zuo1, however, it is not known whether RAC by itself serves as a chaperone for nascent chains. Here we show that, via its rudimentary substrate binding domain (SBD), Ssz1 directly binds to emerging nascent chains prior to Ssb. Structural and biochemical analyses identify a conserved LP-motif at the Zuo1 N-terminus forming a polyproline-II helix, which binds to the Ssz1-SBD as a pseudo-substrate. The LP-motif competes with nascent chain binding to the Ssz1-SBD and modulates nascent chain transfer. The combined data indicate that Ssz1 is an active chaperone optimized for transient, low-affinity substrate binding, which ensures the flux of nascent chains through RAC/Ssb.

MetAP-like Ebp1 occupies the human ribosomal tunnel exit and recruits flexible rRNA expansion segments

Human Ebp1 is a member of the proliferation-associated 2G4 (PA2G4) family and plays an important role in cancer regulation. Ebp1 shares the methionine aminopeptidase (MetAP) fold and binds to mature 80S ribosomes for translational control. Here, we present a cryo-EM single particle analysis reconstruction of Ebp1 bound to non-translating human 80S ribosomes at a resolution range from 3.3 to ~8 Å. Ebp1 blocks the tunnel exit with major interactions to the general uL23/uL29 docking site for nascent chain-associated factors complemented by eukaryote-specific eL19 and rRNA helix H59. H59 is defined as dynamic adaptor undergoing significant remodeling upon Ebp1 binding. Ebp1 recruits rRNA expansion segment ES27L to the tunnel exit via specific interactions with rRNA consensus sequences. The Ebp1-ribosome complex serves as a template for MetAP binding and provides insights into the structural principles for spatial coordination of co-translational events and molecular triage at the ribosomal tunnel exit.

Nascent SecM chain interacts with outer ribosomal surface to stabilize translation arrest.

SecM, a bacterial secretion monitor protein, posttranscriptionally regulates downstream gene expression via translation elongation arrest. SecM contains a characteristic amino acid sequence called the arrest sequence at its C-terminus, and this sequence acts within the ribosomal exit tunnel to stop translation. It has been widely assumed that the arrest sequence within the ribosome tunnel is sufficient for translation arrest. We have previously shown that the nascent SecM chain outside the ribosomal exit tunnel stabilizes translation arrest, but the molecular mechanism is unknown. In this study, we found that residues 57–98 of the nascent SecM chain are responsible for stabilizing translation arrest. We performed alanine/serine-scanning mutagenesis of residues 57–98 to identify D79, Y80, W81, H84, R87, I90, R91, and F95 as the key residues responsible for stabilization. The residues were predicted to be located on and near an α-helix-forming segment. A striking feature of the α-helix is the presence of an arginine patch, which interacts with the negatively charged ribosomal surface. A photocross-linking experiment showed that Y80 is adjacent to the ribosomal protein L23, which is located next to the ribosomal exit tunnel when translation is arrested. Thus, the folded nascent SecM chain that emerges from the ribosome exit tunnel interacts with the outer surface of the ribosome to stabilize translation arrest.