Ribosomal Exit Tunnel Research Articles

NAC controls cotranslational N-terminal methionine excision in eukaryotes.

N-terminal methionine excision from newly synthesized proteins, catalyzed cotranslationally by methionine aminopeptidases (METAPs), is an essential and universally conserved process that plays a key role in cell homeostasis and protein biogenesis. However, how METAPs interact with ribosomes and how their cleavage specificity is ensured is unknown. We discovered that in eukaryotes the nascent polypeptide-associated complex (NAC) controls ribosome binding of METAP1. NAC recruits METAP1 using a long, flexible tail and provides a platform for the formation of an active methionine excision complex at the ribosomal tunnel exit. This mode of interaction ensures the efficient excision of methionine from cytosolic proteins, whereas proteins targeted to the endoplasmic reticulum are spared. Our results suggest a broader mechanism for how access of protein biogenesis factors to translating ribosomes is controlled.

The membrane-binding bacterial toxin long direct repeat D inhibits protein translation

Bacterial plasmids and chromosomes widely contain toxin-antitoxin (TA) loci, which are implicated in stress response, growth regulation and even tolerance to antibiotics and environmental stress. Type I TA systems consist of a stable toxin-expressing mRNA, which is counteracted by an unstable RNA antitoxin. The Long Direct Repeat (LDR-) D locus, a type I TA system of Escherichia Coli (E. coli) K12, encodes a 35 amino acid toxic peptide, LdrD. Despite being characterized as a bacterial toxin, causing rapid killing and nucleoid condensation, little was known about its function and its mechanism of toxicity. Here, we show that LdrD specifically interacts with ribosomes which potentially blocks translation. Indeed, in vitro translation of LdrD-coding mRNA greatly reduces translation efficiency. The structure of LdrD in a hydrophobic environment, similar to the one found in the interior of ribosomes was determined by NMR spectroscopy in 100% trifluoroethanol solution. A single compact α-helix was found which would fit nicely into the ribosomal exit tunnel. Therefore, we conclude that rather than destroying bacterial membranes, LdrD exerts its toxic activity by inhibiting protein synthesis through binding to the ribosomes.

The dynamic architecture of Map1- and NatB-ribosome complexes coordinates the sequential modifications of nascent polypeptide chains.

Cotranslational modification of the nascent polypeptide chain is one of the first events during the birth of a new protein. In eukaryotes, methionine aminopeptidases (MetAPs) cleave off the starter methionine, whereas N-acetyl-transferases (NATs) catalyze N-terminal acetylation. MetAPs and NATs compete with other cotranslationally acting chaperones, such as ribosome-associated complex (RAC), protein targeting and translocation factors (SRP and Sec61) for binding sites at the ribosomal tunnel exit. Yet, whereas well-resolved structures for ribosome-bound RAC, SRP and Sec61, are available, structural information on the mode of ribosome interaction of eukaryotic MetAPs or of the five cotranslationally active NATs is only available for NatA. Here, we present cryo-EM structures of yeast Map1 and NatB bound to ribosome-nascent chain complexes. Map1 is mainly associated with the dynamic rRNA expansion segment ES27a, thereby kept at an ideal position below the tunnel exit to act on the emerging substrate nascent chain. For NatB, we observe two copies of the NatB complex. NatB-1 binds directly below the tunnel exit, again involving ES27a, and NatB-2 is located below the second universal adapter site (eL31 and uL22). The binding mode of the two NatB complexes on the ribosome differs but overlaps with that of NatA and Map1, implying that NatB binds exclusively to the tunnel exit. We further observe that ES27a adopts distinct conformations when bound to NatA, NatB, or Map1, together suggesting a contribution to the coordination of a sequential activity of these factors on the emerging nascent chain at the ribosomal exit tunnel.

The human ribosome-associated complex suppresses prion formation in yeast.

Many human diseases are associated with the misfolding of amyloidogenic proteins. Understanding the mechanisms cells employ to ensure the integrity of the proteome is therefore a crucial step in the development of potential therapeutic interventions. Yeast cells possess numerous prion-forming proteins capable of adopting amyloid conformations, possibly as an epigenetic mechanism to cope with changing environmental conditions. The ribosome-associated complex (RAC), which docks near the ribosomal polypeptide exit tunnel and recruits the Hsp70 Ssb to chaperone nascent chains, can moderate the acquisition of these amyloid conformations in yeast. Here we examine the ability of the human RAC chaperone proteins Mpp11 and Hsp70L1 to function in place of their yeast RAC orthologues Zuo1 and Ssz1 in yeast lacking endogenous RAC and investigate the extent to which the human orthologues can perform RAC chaperone activities in yeast. We found that the Mpp11/Hsp70L1 complex can partially correct the growth defect seen in RAC-deficient yeast cells, although yeast/human hetero species complexes were variable in this ability. The proportion of cells in which the Sup35 protein undergoes spontaneous conversion to a [PSI+ ] prion conformation, which is increased in the absence of RAC, was reduced by the presence of the human RAC complex. However, the toxicity in yeast from expression of a pathogenically expanded polyQ protein was unable to be countered by the human RAC chaperones. This yeast system can serve as a facile model for studying the extent to which the human RAC chaperones contribute to combating cotranslational misfolding of other mammalian disease-associated proteins.

The protein escape process at the ribosomal exit tunnel has conserved mechanisms across the domains of life.

The ribosomal exit tunnel is the primary structure affecting the release of nascent proteins at the ribosome. The ribosomal exit tunnels from different species have elements of conservation and differentiation in structural and physico-chemical properties. In this study, by simulating the elongation and escape processes of nascent proteins at the ribosomal exit tunnels of four different organisms, we show that the escape process has conserved mechanisms across the domains of life. Specifically, it is found that the escape process of proteins follows the diffusion mechanism given by a simple diffusion model, and the median escape time positively correlates with the number of hydrophobic residues and the net charge of a protein for all the exit tunnels considered. These properties hold for 12 distinct proteins considered in two slightly different and improved Gō-like models. It is also found that the differences in physico-chemical properties of the tunnels lead to quantitative differences in the protein escape times. In particular, the relatively strong hydrophobicity of E. coli's tunnel and the unusually high number of negatively charged amino acids on the tunnel's surface of H. marismortui lead to substantially slower escapes of proteins at these tunnels than at those of S. cerevisiae and H. sapiens.

Accumulation of lankamycin derivative with a branched-chain sugar from a blocked mutant of chalcose biosynthesis in Streptomyces rochei 7434AN4

Lankamycin, a macrolide antibiotic produced by Streptomyces rochei 7434AN4, exhibits a moderate antimicrobial activity and acts as a synergistic pair with carbocyclic antibiotic lankacidin C by binding to the ribosome exit tunnel. Its biosynthetic gene (lkm) cluster (orf24-orf53) is located on the largest plasmid pSLA2-L (210,614 bp). Our group possesses a variety of lankamycin derivatives and macrolide-modification enzymes including P450 enzymes and glycosyltransferases, which may lead to expand the chemical library of bioactive macrolides. Here we constructed a mutant of a 3-ketoreductase gene lkmCVI (orf42) involved in d-chalcose biosynthesis, and its metabolite was isolated and structure-elucidated. Accumulation of novel lankamycin derivative harboring a branched-chain deoxysugar, 5-O-(4′,6′-dideoxy-3′-C-acetyl-d-ribo-hexopyranosyl)-3-O-(4″-O-acetyl-l-arcanosyl)-lankanolide, indicated that LkmCVI acts as a gate keeper enzyme for d-chalcose synthesis in lankamycin biosynthesis.

A male germ-cell-specific ribosome controls male fertility.

Ribosomes are highly sophisticated translation machines that have been demonstrated to be heterogeneous in the regulation of protein synthesis1,2. Male germ cell development involves complex translational regulation during sperm formation3. However, it remains unclear whether translation during sperm formation is performed by a specific ribosome. Here we report a ribosome with a specialized nascent polypeptide exit tunnel, RibosomeST, that is assembled with the male germ-cell-specific protein RPL39L, the paralogue of core ribosome (RibosomeCore) protein RPL39. Deletion of RibosomeST in mice causes defective sperm formation, resulting in substantially reduced fertility. Our comparison of single-particle cryo-electron microscopy structures of ribosomes from mouse kidneys and testes indicates that RibosomeST features a ribosomal polypeptide exit tunnel of distinct size and charge states compared with RibosomeCore. RibosomeST predominantly cotranslationally regulates the folding of a subset of male germ-cell-specific proteins that are essential for the formation of sperm. Moreover, we found that specialized functions of RibosomeST were not replaceable by RibosomeCore. Taken together, identification of this sperm-specific ribosome should greatly expand our understanding of ribosome function and tissue-specific regulation of protein expression pattern in mammals.

Geometric differences in the ribosome exit tunnel impact the escape of small nascent proteins

The exit tunnel is the subcompartment of the ribosome that contains the nascent polypeptide chain and, as such, is involved in various vital functions, including regulation of translation and protein folding. As the geometry of the tunnel shows important differences across species, we focus on key geometrical features of eukaryote and prokaryote tunnels. We used a simple coarse-grained molecular dynamics model to study the role of the tunnel geometry in the post-translational escape of short proteins (short open reading frames [sORFs]) with lengths ranging from 6 to 56 amino acids. We found that the probability of escape for prokaryotes is one for all but the 12-mer chains. Moreover, proteins of this length have an extremely low escape probability in eukaryotes. A detailed examination of the associated single trajectories and energy profiles showed that these variations can be explained by the interplay between the protein configurational space and the confinement effects introduced by the constriction sites of the ribosome exit tunnel. For certain lengths, either one or both of the constriction sites can lead to the trapping of the protein in the “pocket” regions preceding these sites. As the distribution of existing sORFs indicates some bias in length that is consistent with our findings, we finally suggest that the constraints imposed by the tunnel geometry have impacted the evolution of sORFs.

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.

Effects of Hydrophobic and Electrostatic Interactions on the Escape of Nascent Proteins at Bacterial Ribosomal Exit Tunnel

We study the escape process of nascent proteins at the ribosomal exit tunnel of bacterial Escherichia coli by using molecular dynamics simulations with coarse-grained and atomistic models. It is shown that the effects of hydrophobic and electrostatic interactions on the protein escape at the E. coli's tunnel are qualitatively similar to those obtained previously at the exit tunnel of archaeal Haloarcula marismortui, despite significant differences in the structures and interactions of the ribosome tunnels from the two organisms. Most proteins escape efficiently and their escape time distributions can be fitted to a simple diffusion model. Attractive interactions between nascent protein and the tunnel can significantly slow down the escape process, as shown for the CI2 protein. Interestingly, it is found that the median escape times of the considered proteins (excluding CI2) strongly correlate with the function \(N_h + 5.9 Q\) of the number of hydrophobic residues, \(N_h\), and the net charge, \(Q\), of a protein, with a correlation coefficient of 0.958 for the E. coli's tunnel. The latter result is in quantitative agreement with a previous result for the H. marismortui's tunnel.

Insights into the ribosome function from the structures of non-arrested ribosome-nascent chain complexes.

During protein synthesis, the growing polypeptide threads through the ribosomal exit tunnel and modulates ribosomal activity by itself or by sensing various small molecules, such as metabolites or antibiotics, appearing in the tunnel. While arrested ribosome-nascent chain complexes (RNCCs) have been extensively studied structurally, the lack of a simple procedure for the large-scale preparation of peptidyl-tRNAs, intermediates in polypeptide synthesis that carry the growing chain, means that little attention has been given to RNCCs representing functionally active states of the ribosome. Here we report the facile synthesis of stably linked peptidyl-tRNAs through a chemoenzymatic approach based on native chemical ligation and use them to determine several structures of RNCCs in the functional pre-attack state of the peptidyl transferase centre. These structures reveal that C-terminal parts of the growing peptides adopt the same uniform β-strand conformation stabilized by an intricate network of hydrogen bonds with the universally conserved 23S rRNA nucleotides, and explain how the ribosome synthesizes growing peptides containing various sequences with comparable efficiencies.

Defective Human SRP Induces Protein Quality Control and Triggers Stress Response

Regulation of Aberrant Protein Production (RAPP) is a protein quality control in mammalian cells. RAPP degrades mRNAs of nascent proteins not able to associate with their natural interacting partners during synthesis at the ribosome. However, little is known about the molecular mechanism of the pathway, its substrates, or its specificity. The Signal Recognition Particle (SRP) is the first interacting partner for secretory proteins. It recognizes signal sequences of the nascent polypeptides when they are exposed from the ribosomal exit tunnel. Here, we reveal the generality of the RAPP pathway on the whole transcriptome level through depletion of human SRP54, an SRP subunit. This depletion triggers RAPP and leads to decreased expression of the mRNAs encoding a number of secretory and membrane proteins. The loss of SRP54 also leads to the dramatic upregulation of a specific network of HSP70/40/90 chaperones (HSPA1A, DNAJB1, HSP90AA1, and others), increased ribosome associated ubiquitination, and change in expression of RPS27 and RPS27L suggesting ribosome rearrangement. These results demonstrate the complex nature of defects in protein trafficking, mRNA and protein quality control, and provide better understanding of their mechanisms at the ribosome.

Application of fluorescence correlation spectroscopy to investigate the dynamics of a ribosome-associated trigger factor in Escherichia coli.

Co-translational protein folding is one of the central topics in molecular biology. In Escherichia coli, trigger factor (TF) is a primary chaperone that facilitates co-translational folding by directly interacting with nascent polypeptide chains on translating ribosomes. In this study, we applied fluorescence correlation spectroscopy (FCS), which can analyze the diffusion properties of fluorescent molecules by measuring the fluctuations of the fluorescent intensity, to investigate the interaction between TF and a nascent chain on translating ribosomes both in vitro and in vivo. The FCS analysis with a reconstituted cell-free translation system revealed that the interaction of fluorescently labeled TF with a nascent chain depended on the emergence of the nascent chain from the ribosome exit tunnel, and this interaction was not inhibited by excess amounts of other chaperones. Furthermore, the translation-dependent interaction between GFP-fused TFs and nascent chains was also observed in living E. coli cells. The FCS-based approach established here could be an effective method to investigate the dynamics of other ribosome-associated chaperones besides TF.

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.

Folding and Evolution of a Repeat Protein on the Ribosome

Life on earth is the result of the work of proteins, the cellular nanomachines that fold into elaborated 3D structures to perform their functions. The ribosome synthesizes all the proteins of the biosphere, and many of them begin to fold during translation in a process known as cotranslational folding. In this work we discuss current advances of this field and provide computational and experimental data that highlight the role of ribosome in the evolution of protein structures. First, we used the sequence of the Ankyrin domain from the Drosophila Notch receptor to launch a deep sequence-based search. With this strategy, we found a conserved 33-residue motif shared by different protein folds. Then, to see how the vectorial addition of the motif would generate a full structure we measured the folding on the ribosome of the Ankyrin repeat protein. Not only the on-ribosome folding data is in full agreement with classical in vitro biophysical measurements but also it provides experimental evidence on how folded proteins could have evolved by duplication and fusion of smaller fragments in the RNA world. Overall, we discuss how the ribosomal exit tunnel could be conceptualized as an active site that is under evolutionary pressure to influence protein folding.

MRNA and tRNA modification states influence ribosome speed and frame maintenance during poly(lysine) peptide synthesis

Ribosome speed is dictated by multiple factors including substrate availability, cellular conditions, and product (peptide) formation. Translation slows during the synthesis of cationic peptide sequences, potentially influencing the expression of thousands of proteins. Available evidence suggests that ionic interactions between positively charged nascent peptides and the negatively charged ribosome exit tunnel impede translation. However, this hypothesis was difficult to test directly because of inability to decouple the contributions of amino acid charge from mRNA sequence and tRNA identity/abundance in cells. Furthermore, it is unclear if other components of the translation system central to ribosome function (e.g., RNA modification) influence the speed and accuracy of positively charged peptide synthesis. In this study, we used a fully reconstituted Escherichia coli translation system to evaluate the effects of peptide charge, mRNA sequence, and RNA modification status on the translation of lysine-rich peptides. Comparison of translation reactions on poly(lysine)-encoding mRNAs conducted with either Lys-tRNALys or Val-tRNALys reveals that that amino acid charge, while important, only partially accounts for slowed translation on these transcripts. We further find that in addition to peptide charge, mRNA sequence and both tRNA and mRNA modification status influence the rates of amino acid addition and the ribosome’s ability to maintain frame (instead of entering the −2, −1, and +1 frames) during poly(lysine) peptide synthesis. Our observations lead us to expand the model for explaining how the ribosome slows during poly(lysine) peptide synthesis and suggest that posttranscriptional RNA modifications can provide cells a mechanism to precisely control ribosome movements along an mRNA.

Oxygen level regulates N-terminal translation elongation of selected proteins through deoxyhypusine hydroxylation.

Hypusine is a post-translational modification on eukaryotic translation initiation factor 5A (eIF5A). The last step of hypusine biosynthesis, deoxyhypusine hydroxylation, is an oxygen-dependent reaction. Here we show that deletion of the deoxyhypusine hydroxylase Lia1 compromises yeast respiration through translation downregulation of selected proteins in the respiration pathway. The translation suppression, because of the lack of deoxyhypusine hydroxylation, mainly affects translation of the N termini of the proteins, independent of the presence of proline residues but likely dependent on the interaction between the N-terminal nascent peptide and the ribosomal peptide exit tunnel. Proteomics and biochemical studies reveal that Lia1 deletion decreases N-terminal translation of proteins involved in mitochondrial respiration, oxidative stress response, and protein folding. Our work uncovers functions of the hypusine modification by considering the substrate requirement of the post-translational modification, highlights the unique challenges of translating the N termini of proteins, and reveals an oxygen-sensing mechanism in eukaryotic cells.

The Ribosome Is the Ultimate Receptor for Trypsin Modulating Oostatic Factor (TMOF).

Aedes aegypti Trypsin Modulating Oostatic Factor (AeaTMOF). a mosquito decapeptide that controls trypsin biosynthesis in female and larval mosquitoes. enters the gut epithelial cells of female mosquitoes using ABC-tmfA receptor/importer. To study the ultimate targeted receptor after AeaTMOF enters the cell, AeaTMOF was incubated in vitro with either Escherichia coli or Spodoptera frugiperda protein-expressing extracts containing 70S and 80S ribosomes, respectively. The effect of AeaTMOF on luciferase biosynthesis in vitro using 70S ribosomes was compared with that of oncocin112 (1–13), a ribosome-binding antibacterial peptide. The IC50 of 1 μM and 2 μM, respectively, for both peptides was determined. Incubation with a protein-expressing system and S. frugiperda 80S ribosomes determined an IC50 of 1.8 μM for Aedes aegypti larval late trypsin biosynthesis. Incubation of purified E. coli ribosome with increasing concentration of AeaTMOF shows that the binding of AeaTMOF to the bacterial ribosome exhibits a high affinity (KD = 23 ± 3.4 nM, Bmax = 0.553 ± 0.023 pmol/μg ribosome and Kassoc = 4.3 × 107 M−1). Molecular modeling and docking experiments show that AeaTMOF binds bacterial and Drosophila ribosome (50S and 60S, respectively) at the entrance of the ribosome exit tunnel, blocking the tRNA entrance and preventing protein biosynthesis. Recombinant E. coli cells that express only ABC-tmfA importer are inhibited by AeaTMOF but not by oncocin112 (1–13). These results suggest that the ribosome is the ultimate targeted receptor of AeaTMOF.

From Alpha to Beta – a co-translational way to fold?

ABSTRACT Protein folding in the cell is largely a co-translational process occurring during protein synthesis on the ribosome. It has become evident that co-translational folding is characteristic to almost every protein in the cell of pro- and eukaryotic origin that are single and multidomain, single and multisubunit, cytosolic, secretory and membrane. Co-translational protein folding begins very early during the process of polypeptide chain synthesis on the ribosome, with some secondary structure elements forming inside the ribosomal tunnel and some tertiary structures forming inside the vestibule (lower/wider) region of the ribosomal exit tunnel. However, many details of co-translational folding remains incompletely understood. New data show that folding of a β-barrel protein begins with formation of an α-helix inside the ribosome that rearranges into a β-hairpin structure as the growing peptide reaches the wider/vestibule region of the exit tunnel. While it was previously suggested that such scenario can take place on the ribosome, the new data provide the first experimental evidence in support of this notion.

The Identity of the Constriction Region of the Ribosomal Exit Tunnel Is Important to Maintain Gene Expression in Escherichia coli.

ABSTRACTMutational changes in bacterial ribosomes often affect gene expression and consequently cellular fitness. Understanding how mutant ribosomes disrupt global gene expression is critical to determining key genetic factors that affect bacterial survival. Here, we describe gene expression and phenotypic changes presented in Escherichia coli cells carrying an uL22(K90D) mutant ribosomal protein, which displayed alterations during growth. Ribosome profiling analyses revealed reduced expression of operons involved in catabolism, indole production, and lysine-dependent acid resistance. In general, translation initiation of proximal genes in several of these affected operons was substantially reduced. These reductions in expression were accompanied by increases in the expression of acid-induced membrane proteins and chaperones, the glutamate-decarboxylase regulon, and the autoinducer-2 metabolic regulon. In agreement with these changes, uL22(K90D) mutant cells had higher glutamate decarboxylase activity, survived better in extremely acidic conditions, and generated more biofilm in static cultures compared to their parental strain. Our work demonstrates that a single mutation in a non-conserved residue of a ribosomal protein affects a substantial number of genes to alter pH resistance and the formation of biofilms.IMPORTANCE All newly synthesized proteins must pass through a channel in the ribosome named the exit tunnel before emerging into the cytoplasm, membrane, and other compartments. The structural characteristics of the tunnel could govern protein folding and gene expression in a species-specific manner but how the identity of tunnel elements influences gene expression is less well-understood. Our global transcriptomics and translatome profiling demonstrate that a single substitution in a non-conserved amino acid of the E. coli tunnel protein uL22 has a profound impact on catabolism, cellular signaling, and acid resistance systems. Consequently, cells bearing the uL22 mutant ribosomes had an increased ability to survive acidic conditions and form biofilms. This work reveals a previously unrecognized link between tunnel identity and bacterial stress adaptation involving pH response and biofilm formation.