Ribosomal Surface Research Articles

Reduction of Ribosomal Expansion Segments in Yeast Species of the Magnusiomyces/Saprochaete Clade.

Ribosomes are ribonucleoprotein complexes highly conserved across all domains of life. The size differences of ribosomal RNAs (rRNAs) can be mainly attributed to variable regions termed expansion segments (ESs) protruding out from the ribosomal surface. The ESs were found to be involved in a range of processes including ribosome biogenesis and maturation, translation, and co-translational protein modification. Here, we analyze the rRNAs of the yeasts from the Magnusiomyces/Saprochaete clade belonging to the basal lineages of the subphylum Saccharomycotina. We find that these yeasts are missing more than 400 nt from the 25S rRNA and 150 nt from the 18S rRNAs when compared to their canonical counterparts in Saccharomyces cerevisiae. The missing regions mostly map to ESs, thus representing a shift toward a minimal rRNA structure. Despite the structural changes in rRNAs, we did not identify dramatic alterations in the ribosomal protein inventories. We also show that the size-reduced rRNAs are not limited to the species of the Magnusiomyces/Saprochaete clade, indicating that the shortening of ESs happened independently in several other lineages of the subphylum Saccharomycotina.

Nascent chains derived from a foldable protein sequence interact with specific ribosomal surface sites near the exit tunnel

In order to become bioactive, proteins must be translated and protected from aggregation during biosynthesis. The ribosome and molecular chaperones play a key role in this process. Ribosome-bound nascent chains (RNCs) of intrinsically disordered proteins and RNCs bearing a signal/arrest sequence are known to interact with ribosomal proteins. However, in the case of RNCs bearing foldable protein sequences, not much information is available on these interactions. Here, via a combination of chemical crosslinking and time-resolved fluorescence-anisotropy, we find that nascent chains of the foldable globin apoHmp1–140 interact with ribosomal protein L23 and have a freely-tumbling non-interacting N-terminal compact region comprising 63–94 residues. Longer RNCs (apoHmp1–189) also interact with an additional yet unidentified ribosomal protein, as well as with chaperones. Surprisingly, the apparent strength of RNC/r-protein interactions does not depend on nascent-chain sequence. Overall, foldable nascent chains establish and expand interactions with selected ribosomal proteins and chaperones, as they get longer. These data are significant because they reveal the interplay between independent conformational sampling and nascent-protein interactions with the ribosomal surface.

Cryo-EMstructure of SRP68/72 reveals an extended dimerization domain with RNA-binding activity.

The signal recognition particle (SRP) is a critical component in protein sorting pathways in all domains of life. Human SRP contains six proteins bound to the 7S RNA and their structures and functions have been mostly elucidated. The SRP68/72 dimer is the largest SRP component and is essential for SRP function. Although the structures of the SRP68/72 RNA binding and dimerization domains have been previously reported, the structure and function of large portions of the SRP68/72 dimer remain unknown. Here, we analyse full-length SRP68/72 using cryo-EM and report that SRP68/72 depend on each other for stability and form an extended dimerization domain. This newly observed dimerization domain is both a protein- and RNA-binding domain. Comparative analysis with current structural models suggests that this dimerization domain undergoes dramatic translocation upon SRP docking onto SRP receptor and eventually comes close to the Alu domain. We propose that the SRP68/72 dimerization domain functions by binding and detaching the Alu domain and SRP9/14 from the ribosomal surface, thus releasing elongation arrest upon docking onto the ER membrane.

A Conserved Ribosomal Protein Has Entirely Dissimilar Structures in Different Organisms.

Ribosomes from different species can markedly differ in their composition by including dozens of ribosomal proteins that are unique to specific lineages but absent in others. However, it remains unknown how ribosomes acquire new proteins throughout evolution. Here, to help answer this question, we describe the evolution of the ribosomal protein msL1/msL2 that was recently found in ribosomes from the parasitic microorganism clade, microsporidia. We show that this protein has a conserved location in the ribosome but entirely dissimilar structures in different organisms: in each of the analyzed species, msL1/msL2 exhibits an altered secondary structure, an inverted orientation of the N-termini and C-termini on the ribosomal binding surface, and a completely transformed 3D fold. We then show that this fold switching is likely caused by changes in the ribosomal msL1/msL2-binding site, specifically, by variations in rRNA. These observations allow us to infer an evolutionary scenario in which a small, positively charged, de novo-born unfolded protein was first captured by rRNA to become part of the ribosome and subsequently underwent complete fold switching to optimize its binding to its evolving ribosomal binding site. Overall, our work provides a striking example of how a protein can switch its fold in the context of a complex biological assembly, while retaining its specificity for its molecular partner. This finding will help us better understand the origin and evolution of new protein components of complex molecular assemblies-thereby enhancing our ability to engineer biological molecules, identify protein homologs, and peer into the history of life on Earth.

Folding stabilities of ribosome-bound nascent polypeptides probed by mass spectrometry

The folding of most proteins occurs during the course of their translation while their tRNA-bound C termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry-based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurement subglobal folding stabilities of ribosome nascent chains within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (dihydrofolate reductase, chemotaxis protein Y, and DNA polymerase IV) in soluble and ribosome-bound states. The data indicate that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.

Systematic genetic identification of functional domains on collided di-ribosomes responsible for rescue pathways upon translation arrest in Saccharomyces cerevisiae.

Translation elongation becomes arrested when various obstacles arise, such as a series of inefficient rare codons or stable RNA secondary structures, thus causing ribosomal stalling along the mRNA. Certain wasteful and persistent stalling states are resolved by ribosome rescue pathways. For instance, collisions between stalled and subsequent ribosomes are thought to induce ubiquitination of ribosomal S20 protein by the E3 ubiquitin ligase Hel2, which triggers subsequent rescue reactions. Although structural studies have revealed specific contact sites between collided ribosomes, theribosomal regions crucial for the rescue reaction remain uncharacterized. In this study, we performed a systematic genetic analysis to identifythe molecular regions required for ribosome rescue in Saccharomyces cerevisiae. A series of dominant negative mutations capable of abolishing the rescue reaction were isolated in ribosomal proteins S20 and Asc1. Moreover, mutations in both proteins clustered on the surface of ribosomes between the collided ribosome interfaces, aligned in such a way that they seemingly faced each other. Further analysis via the application of the split-TRP1 protein assay revealed that the mutation of either protein distinctively affected the functional interaction between Hel2 and Asc1, suggesting the development of differential functionality at the interface between collided ribosomes. Our results provide novel and complementary insights into the detailed molecular mechanisms of ribosomal rescue pathways.

Binding of the peptide deformylase on the ribosome surface modulates the exit tunnel interior

Proteosynthesis on ribosomes is regulated at many levels. Conformational changes of the ribosome, possibly induced by external factors, may transfer over large distances and contribute to the regulation. The molecular principles of this long-distance allostery within the ribosome remain poorly understood. Here, we use structural analysis and atomistic molecular dynamics simulations to investigate peptide deformylase (PDF), an enzyme that binds to the ribosome surface near the ribosomal protein uL22 during translation and chemically modifies the emerging nascent peptide. Our simulations of the entire ribosome-PDF complex reveal that the PDF undergoes a swaying motion on the ribosome surface at the submicrosecond timescale. We show that the PDF affects the conformational dynamics of parts of the ribosome over distances of more than 5 nm. Using a supervised-learning algorithm, we demonstrate that the exit tunnel is influenced by the presence or absence of PDF. Our findings suggest a possible effect of the PDF on the nascent peptide translocation through the ribosome exit tunnel.

Modulating co-translational protein folding by rational design and ribosome engineering

Co-translational folding is a fundamental process for the efficient biosynthesis of nascent polypeptides that emerge through the ribosome exit tunnel. To understand how this process is modulated by the shape and surface of the narrow tunnel, we have rationally engineered three exit tunnel protein loops (uL22, uL23 and uL24) of the 70S ribosome by CRISPR/Cas9 gene editing, and studied the co-translational folding of an immunoglobulin-like filamin domain (FLN5). Our thermodynamics measurements employing 19F/15N/methyl-TROSY NMR spectroscopy together with cryo-EM and molecular dynamics simulations reveal how the variations in the lengths of the loops present across species exert their distinct effects on the free energy of FLN5 folding. A concerted interplay of the uL23 and uL24 loops is sufficient to alter co-translational folding energetics, which we highlight by the opposite folding outcomes resulting from their extensions. These subtle modulations occur through a combination of the steric effects relating to the shape of the tunnel, the dynamic interactions between the ribosome surface and the unfolded nascent chain, and its altered exit pathway within the vestibule. These results illustrate the role of the exit tunnel structure in co-translational folding, and provide principles for how to remodel it to elicit a desired folding outcome.

Ribosome-nascent Chain Interaction Regulates N-terminal Protein Modification

Numerous proteins initiate their folding, localization, and modifications early during translation, and emerging data show that the ribosome actively participates in diverse protein biogenesis pathways. Here we show that the ribosome imposes an additional layer of substrate selection during N-terminal methionine excision (NME), an essential protein modification in bacteria. Biochemical analyses show that cotranslational NME is exquisitely sensitive to a hydrophobic signal sequence or transmembrane domain near the N terminus of the nascent polypeptide. The ability of the nascent chain to access the active site of NME enzymes dictates NME efficiency, which is inhibited by confinement of the nascent chain on the ribosome surface and exacerbated by signal recognition particle. In vivo measurements corroborate the inhibition of NME by an N-terminal hydrophobic sequence, suggesting the retention of formylmethionine on a substantial fraction of the secretory and membrane proteome. Our work demonstrates how molecular features of a protein regulate its cotranslational modification and highlights the active participation of the ribosome in protein biogenesis pathways via interactions of the ribosome surface with the nascent protein.

Common sequence motifs of nascent chains engage the ribosome surface and trigger factor

In the cell, the conformations of nascent polypeptide chains during translation are modulated by both the ribosome and its associated molecular chaperone, trigger factor. The specific interactions that underlie these modulations, however, are still not known in detail. Here, we combine protein engineering, in-cell and invitro NMR spectroscopy, and molecular dynamics simulations to explore how proteins interact with the ribosome during their biosynthesis before folding occurs. Our observations of α-synuclein nascent chains in living Escherichia coli cells reveal that ribosome surface interactions dictate the dynamics of emerging disordered polypeptides in the crowded cytosol. We show that specific basic and aromatic motifs drive such interactions and directly compete with trigger factor binding while biasing the direction of the nascent chain during its exit out of the tunnel. These results reveal a structural basis for the functional role of the ribosome as a scaffold with holdase characteristics and explain how handover of the nascent chain to specific auxiliary proteins occurs among a host of other factors in the cytosol.

An intrinsically disordered nascent protein interacts with specific regions of the ribosomal surface near the exit tunnel

The influence of the ribosome on nascent chains is poorly understood, especially in the case of proteins devoid of signal or arrest sequences. Here, we provide explicit evidence for the interaction of specific ribosomal proteins with ribosome-bound nascent chains (RNCs). We target RNCs pertaining to the intrinsically disordered protein PIR and a number of mutants bearing a variable net charge. All the constructs analyzed in this work lack N-terminal signal sequences. By a combination chemical crosslinking and Western-blotting, we find that all RNCs interact with ribosomal protein L23 and that longer nascent chains also weakly interact with L29. The interacting proteins are spatially clustered on a specific region of the large ribosomal subunit, close to the exit tunnel. Based on chain-length-dependence and mutational studies, we find that the interactions with L23 persist despite drastic variations in RNC sequence. Importantly, we also find that the interactions are highly Mg+2-concentration-dependent. This work is significant because it unravels a novel role of the ribosome, which is shown to engage with the nascent protein chain even in the absence of signal or arrest sequences.

Interactions between nascent proteins and the ribosome surface inhibit co-translational folding

Most proteins begin to fold during biosynthesis on the ribosome. It has been suggested that interactions between the emerging polypeptide and the ribosome surface might allow the ribosome itself to modulate co-translational folding. Here we combine protein engineering and NMR spectroscopy to characterize a series of interactions between the ribosome surface and unfolded nascent chains of the immunoglobulin-like FLN5 filamin domain. The strongest interactions are found for a C-terminal segment that is essential for folding, and we demonstrate quantitative agreement between the strength of this interaction and the energetics of the co-translational folding process itself. Mutations in this region that reduce the extent of binding result in a shift in the co-translational folding equilibrium towards the native state. Our results therefore demonstrate that a competition between folding and binding provides a simple, dynamic mechanism for the modulation of co-translational folding by the ribosome.

Evolutionary and functional relationships in the ribosome biogenesis SBDS and EFL1 protein families.

Nascent ribosomal 60S subunits undergo the last maturation steps in the cytoplasm. The last one involves removing the anti-association factor eIF6 from the 60S ribosomal surface by the joint action of the Elongation Factor-like 1 (EFL1) GTPase and the SBDS protein. Herein, we studied the evolutionary relationship of the EFL1 and EF-2 protein families and the functional conservation within EFL1 orthologues. Phylogenetic analysis demonstrated that the EFL1 proteins are exclusive of eukaryotes and share an evolutionary origin with the EF-2 and EF-G protein families. EFL1 proteins originated by gene duplication from the EF-2 proteins and specialized in ribosome maturation while the latter retained their function in translation. Some organisms have more than one EFL1 protein resulting from alternative splicing, while others are encoded in different genes originated by gene duplication. However, the function of these alternative EFL1 proteins is still unknown. We performed GTPase activity and complementation assays to study the functional conservation of EFL1 homologs alone and together with their SBDS counterparts. None of the orthologues or cross-species combinations could replace the function of the corresponding yeast EFL1•SBDS binomial. The complementation of SBDS interspecies chimeras indicates that domain 2 is vital for its function together with EFL1 and the 60S subunit. The results suggest a functional species-specificity and possible co-evolution between EFL1, SBDS, and the 60S ribosomal subunit. These findings set the basis for further studies directed to understand the molecular evolution of these proteins and their impact on ribosome biogenesis and disease.

Ribosome heterogeneity in Drosophila melanogaster gonads through paralog-switching

Ribosomes have long been thought of as homogeneous macromolecular machines, but recent evidence suggests they are heterogeneous and could be specialised to regulate translation. Here, we have characterised ribosomal protein heterogeneity across 4 tissues of Drosophila melanogaster. We find that testes and ovaries contain the most heterogeneous ribosome populations, which occurs through a combination of paralog-enrichment and paralog-switching. We have solved structures of ribosomes purified from in vivo tissues by cryo-EM, revealing differences in precise ribosomal arrangement for testis and ovary 80S ribosomes. Differences in the amino acid composition of paralog pairs and their localisation on the ribosome exterior indicate paralog-switching could alter the ribosome surface, enabling different proteins to regulate translation. One testis-specific paralog-switching pair is also found in humans, suggesting this is a conserved site of ribosome heterogeneity. Overall, this work allows us to propose that mRNA translation might be regulated in the gonads through ribosome heterogeneity, providing a potential means of ribosome specialisation.

Overlapping regions of Caf20 mediate its interactions with the mRNA-5\u2032cap-binding protein eIF4E and with ribosomes

By interacting with the mRNA 5′ cap, the translation initiation factor eIF4E plays a critical role in selecting mRNAs for protein synthesis in eukaryotic cells. Caf20 is a member of the family of proteins found across eukaryotes termed 4E-BPs, which compete with eIF4G for interaction with eIF4E. Caf20 independently interacts with ribosomes. Thus, Caf20 modulates the mRNA selection process via poorly understood mechanisms. Here we performed unbiased mutagenesis across Caf20 to characterise which regions of Caf20 are important for interaction with eIF4E and with ribosomes. Caf20 binding to eIF4E is entirely dependent on a canonical motif shared with other 4E-BPs. However, binding to ribosomes is weakened by mutations throughout the protein, suggesting an extended binding interface that partially overlaps with the eIF4E-interaction region. By using chemical crosslinking, we identify a potential ribosome interaction region on the ribosome surface that spans both small and large subunits and is close to a known interaction site of eIF3. The function of ribosome binding by Caf20 remains unclear.

The Archaeal Elongation Factor EF-2 Induces the Release of aIF6 From 50S Ribosomal Subunit.

The translation factor IF6 is a protein of about 25 kDa shared by the Archaea and the Eukarya but absent in Bacteria. It acts as a ribosome anti-association factor that binds to the large subunit preventing the joining to the small subunit. It must be released from the large ribosomal subunit to permit its entry to the translation cycle. In Eukarya, this process occurs by the coordinated action of the GTPase Efl1 and the docking protein SBDS. Archaea do not possess a homolog of the former factor while they have a homolog of SBDS. In the past, we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homolog (aIF6) highlighting its similarity to the eukaryotic counterpart. Here, we analyzed the mechanism of aIF6 release from the large ribosomal subunit. We found that, similarly to the Eukarya, the detachment of aIF6 from the 50S subunit requires a GTPase activity which involves the archaeal elongation factor 2 (aEF-2). However, the release of aIF6 from the 50S subunits does not require the archaeal homolog of SBDS, being on the contrary inhibited by its presence. Molecular modeling, using published structural data of closely related homologous proteins, elucidated the mechanistic interplay between the aIF6, aSBDS, and aEF2 on the ribosome surface. The results suggest that a conformational rearrangement of aEF2, upon GTP hydrolysis, promotes aIF6 ejection. On the other hand, aSBDS and aEF2 share the same binding site, whose occupation by SBDS prevents aEF2 binding, thereby inhibiting aIF6 release.

Optimal design of adaptively sampled NMR experiments for measurement of methyl group dynamics with application to a ribosome-nascent chain complex

NMR measurements of cross-correlated nuclear spin relaxation provide powerful probes of polypeptide dynamics and rotational diffusion, free from contributions due to chemical exchange or interactions with external spins. Here, we report on the development of a sensitivity-optimized pulse sequence for the analysis of the differential relaxation of transitions within isolated 13CH3 spin systems, in order to characterise rotational diffusion and side chain order through the product S2τc. We describe the application of optimal design theory to implement a real-time ‘on-the-fly’ adaptive sampling scheme that maximizes the accuracy of the measured parameters. The increase in sensitivity obtained using this approach enables quantitative measurements of rotational diffusion within folded states of translationally-arrested ribosome–nascent chain complexes of the FLN5 filamin domain, and can be used to place strong limits on interactions between the domain and the ribosome surface.

Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit

Mitoribosomes consist of ribosomal RNA and protein components, coordinated assembly of which is critical for function. We used mitoribosomes from Trypanosoma brucei with reduced RNA and increased protein mass to provide insights into the biogenesis of the mitoribosomal large subunit. Structural characterization of a stable assembly intermediate revealed 22 assembly factors, some of which have orthologues/counterparts/homologues in mammalian genomes. These assembly factors form a protein network that spans a distance of 180 Å, shielding the ribosomal RNA surface. The central protuberance and L7/L12 stalk are not assembled entirely and require removal of assembly factors and remodeling of the mitoribosomal proteins to become functional. The conserved proteins GTPBP7 and mt‐EngA are bound together at the subunit interface in proximity to the peptidyl transferase center. A mitochondrial acyl‐carrier protein plays a role in docking the L1 stalk, which needs to be repositioned during maturation. Additional enzymatically deactivated factors scaffold the assembly while the exit tunnel is blocked. Together, this extensive network of accessory factors stabilizes the immature sites and connects the functionally important regions of the mitoribosomal large subunit.

Interaction Between Ribosome-Bound Nascent Proteins and a Specific Region of the Ribosomal Surface

Interaction Between Ribosome-Bound Nascent Proteins and a Specific Region of the Ribosomal Surface

Nascent chain dynamics and ribosome interactions within folded ribosome-nascent chain complexes observed by NMR spectroscopy.

The folding of many proteins can begin during biosynthesis on the ribosome and can be modulated by the ribosome itself. Such perturbations are generally believed to be mediated through interactions between the nascent chain and the ribosome surface, but despite recent progress in characterising interactions of unfolded states with the ribosome, and their impact on the initiation of co-translational folding, a complete quantitative analysis of interactions across both folded and unfolded states of a nascent chain has yet to be realised. Here we apply solution-state NMR spectroscopy to measure transverse proton relaxation rates for methyl groups in folded ribosome–nascent chain complexes of the FLN5 filamin domain. We observe substantial increases in relaxation rates for the nascent chain relative to the isolated domain, which can be related to changes in effective rotational correlation times using measurements of relaxation and cross-correlated relaxation in the isolated domain. Using this approach, we can identify interactions between the nascent chain and the ribosome surface, driven predominantly by electrostatics, and by measuring the change in these interactions as the subsequent FLN6 domain emerges, we may deduce their impact on the free energy landscapes associated with the co-translational folding process.