Architecture of Microcin B17 Synthetase: An Octameric Protein Complex Converting a Ribosomally Synthesized Peptide into a DNA Gyrase Poison.

The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.

Activation of the Stringent Response by Loading of RelA-tRNA Complexes at the Ribosomal A-Site.

RelA/SpoT homologs (RSHs) are ubiquitous bacterial enzymes that synthesize and hydrolyze (p)ppGpp in response to environmental challenges. Bacteria cannot survive in hosts and produce infection without activating the (p)ppGpp-mediated stringent response, but it is not yet understood how the enzymatic activities of RSHs are controlled. Using UV crosslinking and deep sequencing, we show that Escherichia coli RelA ((p)ppGpp synthetase I) interacts with uncharged tRNA without being activated. Amino acid starvation leads to loading of cognate tRNA⋅RelA complexes at vacant ribosomal A-sites. In turn, RelA is activated and synthesizes (p)ppGpp. Mutation of a single, conserved residue in RelA simultaneously prevents tRNA binding, ribosome binding, and activation of RelA, showing that all three processes are interdependent. Our results support a model in which (p)ppGpp synthesis occurs by ribosome-bound RelA interacting with the Sarcin-Ricin loop of 23S rRNA.

ZNF598 and RACK1 Regulate Mammalian Ribosome-Associated Quality Control Function by Mediating Regulatory 40S Ribosomal Ubiquitylation.

Ribosomes that experience terminal stalls during translation are resolved by ribosome-associated quality control (QC) pathways that oversee mRNA and nascent chain destruction and recycle ribosomal subunits. The proximal factors that sense stalled ribosomes and initiate mammalian ribosome-associated QC events remain undefined. We demonstrate that the ZNF598 ubiquitin ligase and the 40S ribosomal protein RACK1 help to resolve poly(A)-induced stalled ribosomes. They accomplish this by regulating distinct and overlapping regulatory 40S ribosomal ubiquitylation events. ZNF598 primarily mediates regulatory ubiquitylation of RPS10 and RPS20, whereas RACK1 regulates RPS2, RPS3, and RPS20 ubiquitylation. Gain or loss of ZNF598 function or mutations that block RPS10 or RPS20 ubiquitylation result in defective resolution of stalled ribosomes and subsequent readthrough of poly(A)-containing stall sequences. Together, our results indicate that ZNF598, RACK1, and 40S regulatory ubiquitylation plays a pivotal role in mammalian ribosome-associated QC pathways.

Transcriptome-wide Analysis of Roles for tRNA Modifications in Translational Regulation.

Covalent nucleotide modifications in noncoding RNAs affect a plethora of biological processes, and new functions continue to be discovered even for well-known modifying enzymes. To systematically compare the functions of a large set of noncoding RNA modifications in gene regulation, we carried out ribosome profiling in budding yeast to characterize 57 nonessential genes involved in tRNA modification. Deletion mutants exhibited a range of translational phenotypes, with enzymes known to modify anticodons, or non-tRNA substrates such as rRNA, exhibiting the most dramatic translational perturbations. Our data build on prior reports documenting translational upregulation of the nutrient-responsive transcription factor Gcn4 in response to numerous tRNA perturbations, and identify many additional translationally regulated mRNAs throughout the yeast genome. Our data also uncover unexpected roles for tRNA-modifying enzymes in regulation of TY retroelements, and in rRNA 2'-O-methylation. This dataset should provide a rich resource for discovery of additional links between tRNA modifications and gene regulation.

The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells.

METTL3 is an RNA methyltransferase implicated in mRNA biogenesis, decay, and translation control through N(6)-methyladenosine (m(6)A) modification. Here we find that METTL3 promotes translation of certain mRNAs including epidermal growth factor receptor (EGFR) and the Hippo pathway effector TAZ in human cancer cells. In contrast to current models that invoke m(6)A reader proteins downstream of nuclear METTL3, we find METTL3 associates with ribosomes and promotes translation in the cytoplasm. METTL3 depletion inhibits translation, and both wild-type and catalytically inactive METTL3 promote translation when tethered to a reporter mRNA. Mechanistically, METTL3 enhances mRNA translation through an interaction with the translation initiation machinery. METTL3 expression is elevated in lung adenocarcinoma and using both loss- and gain-of-function studies, we find that METTL3 promotes growth, survival, and invasion of human lung cancer cells. Our results uncover an important role of METTL3 in promoting translation of oncogenes in human lung cancer.

Amazing Diversity in Biochemical Roles of Fe(II)/2-Oxoglutarate Oxygenases.

Since their discovery in the 1960s, the family of Fe(II)/2-oxoglutarate-dependent oxygenases has undergone a tremendous expansion to include enzymes catalyzing a vast diversity of biologically important reactions. Recent examples highlight roles in controlling chromatin modification, transcription, mRNA demethylation, and mRNA splicing. Others generate modifications in tRNA, translation factors, ribosomes, and other proteins. Thus, oxygenases affect all components of molecular biology's central dogma, in which information flows from DNA to RNA to proteins. These enzymes also function in biosynthesis and catabolism of cellular metabolites, including antibiotics and signaling molecules. Due to their critical importance, ongoing efforts have targeted family members for the development of specific therapeutics. This review provides a general overview of recently characterized oxygenase reactions and their key biological roles.

The pathway to GTPase activation of elongation factor SelB on the ribosome.

In all domains of life, selenocysteine (Sec) is delivered to the ribosome by selenocysteine-specific tRNA (tRNASec) with the help of a specialized translation factor, SelB in bacteria. Sec-tRNASec recodes a UGA stop codon next to a downstream mRNA stem-loop. Here we present the structures of six intermediates on the pathway of UGA recoding in Escherichia coli by single-particle cryo-electron microscopy. The structures explain the specificity of Sec-tRNASec binding by SelB and show large-scale rearrangements of Sec-tRNASec. Upon initial binding of SelB-Sec-tRNASec to the ribosome and codon reading, the 30S subunit adopts an open conformation with Sec-tRNASec covering the sarcin-ricin loop (SRL) on the 50S subunit. Subsequent codon recognition results in a local closure of the decoding site, which moves Sec-tRNASec away from the SRL and triggers a global closure of the 30S subunit shoulder domain. As a consequence, SelB docks on the SRL, activating the GTPase of SelB. These results reveal how codon recognition triggers GTPase activation in translational GTPases.

Splicing-factor oncoprotein SRSF1 stabilizes p53 via RPL5 and induces cellular senescence.

Splicing and translation are highly regulated steps of gene expression. Altered expression of proteins involved in these processes can be deleterious. Therefore, the cell has many safeguards against such misregulation. We report that the oncogenic splicing factor SRSF1, which is overexpressed in many cancers, stabilizes the tumor suppressor protein p53 by abrogating its MDM2-dependent proteasomal degradation. We show that SRSF1 is a necessary component of an MDM2/ribosomal protein complex, separate from the ribosome, that functions in a p53-dependent ribosomal-stress checkpoint pathway. Consistent with the stabilization of p53, increased SRSF1 expression in primary human fibroblasts decreases cellular proliferation and ultimately triggers oncogene-induced senescence (OIS). These findings underscore the deleterious outcome of SRSF1 overexpression and identify a cellular defense mechanism against its aberrant function. Furthermore, they implicate the RPL5-MDM2 complex in OIS and demonstrate a link between spliceosomal and ribosomal components, functioning independently of their canonical roles, to monitor cellular physiology and cell-cycle progression.

Ribosomal oxygenases are structurally conserved from prokaryotes to humans.

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N(ε)-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.

Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super-expression system for structural studies.

The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.

Deciphering Determinants in Ribosomal Methyltransferases That Confer Antimicrobial Resistance.

Post-translational methylation of rRNA at select positions is a prevalent resistance mechanism adopted by pathogens. In this work, KsgA, a housekeeping ribosomal methyltransferase (rMtase) involved in ribosome biogenesis, was exploited as a model system to delineate the specific targeting determinants that impart substrate specificity to rMtases. With a combination of evolutionary and structure-guided approaches, a set of chimeras were created that altered the targeting specificity of KsgA such that it acted similarly to erythromycin-resistant methyltransferases (Erms), rMtases found in multidrug-resistant pathogens. The results revealed that specific loop embellishments on the basic Rossmann fold are key determinants in the selection of the cognate RNA. Moreover, in vivo studies confirmed that chimeric constructs are competent in imparting macrolide resistance. This work explores the factors that govern the emergence of resistance and paves the way for the design of specific inhibitors useful in reversing antibiotic resistance.

Nascent peptide in the ribosome exit tunnel affects functional properties of the A-site of the peptidyl transferase center.

The ability to monitor the nascent peptide structure and to respond functionally to specific nascent peptide sequences is a fundamental property of the ribosome. An extreme manifestation of such response is nascent peptide-dependent ribosome stalling, involved in the regulation of gene expression. The molecular mechanisms of programmed translation arrest are unclear. By analyzing ribosome stalling at the regulatory cistron of the antibiotic resistance gene ermA, we uncovered a carefully orchestrated cooperation between the ribosomal exit tunnel and the A-site of the peptidyl transferase center (PTC) in halting translation. The presence of an inducing antibiotic and a specific nascent peptide in the exit tunnel abrogate the ability of the PTC to catalyze peptide bond formation with a particular subset of amino acids. The extent of the conferred A-site selectivity is modulated by the C-terminal segment of the nascent peptide, where the third-from-last residue plays a critical role.

The emerging roles of ribosomal histidyl hydroxylases in cell biology, physiology and disease.

Hydroxylation is a novel protein modification catalyzed by a family of oxygenases that depend on fundamental nutrients and metabolites for activity. Protein hydroxylases have been implicated in a variety of key cellular processes that play important roles in both normal homeostasis and pathogenesis. Here, in this review, we summarize the current literature on a highly conserved sub-family of oxygenases that catalyze protein histidyl hydroxylation. We discuss the evidence supporting the biochemical assignment of these emerging enzymes as ribosomal protein hydroxylases, and provide an overview of their role in immunology, bone development, and cancer.

Evolution of Robustness to Protein Mistranslation by Accelerated Protein Turnover.

Translational errors occur at high rates, and they influence organism viability and the onset of genetic diseases. To investigate how organisms mitigate the deleterious effects of protein synthesis errors during evolution, a mutant yeast strain was engineered to translate a codon ambiguously (mistranslation). It thereby overloads the protein quality-control pathways and disrupts cellular protein homeostasis. This strain was used to study the capacity of the yeast genome to compensate the deleterious effects of protein mistranslation. Laboratory evolutionary experiments revealed that fitness loss due to mistranslation can rapidly be mitigated. Genomic analysis demonstrated that adaptation was primarily mediated by large-scale chromosomal duplication and deletion events, suggesting that errors during protein synthesis promote the evolution of genome architecture. By altering the dosages of numerous, functionally related proteins simultaneously, these genetic changes introduced large phenotypic leaps that enabled rapid adaptation to mistranslation. Evolution increased the level of tolerance to mistranslation through acceleration of ubiquitin-proteasome-mediated protein degradation and protein synthesis. As a consequence of rapid elimination of erroneous protein products, evolution reduced the extent of toxic protein aggregation in mistranslating cells. However, there was a strong evolutionary trade-off between adaptation to mistranslation and survival upon starvation: the evolved lines showed fitness defects and impaired capacity to degrade mature ribosomes upon nutrient limitation. Moreover, as a response to an enhanced energy demand of accelerated protein turnover, the evolved lines exhibited increased glucose uptake by selective duplication of hexose transporter genes. We conclude that adjustment of proteome homeostasis to mistranslation evolves rapidly, but this adaptation has several side effects on cellular physiology. Our work also indicates that translational fidelity and the ubiquitin-proteasome system are functionally linked to each other and may, therefore, co-evolve in nature.

Distribution of the ribosome associated endonuclease Rae1 and the potential role of conserved amino acids in codon recognition.

We recently identified a novel ribonuclease in Bacillus subtilis called Rae1 that cleaves mRNAs in a translation-dependent manner. Rae1 is a member of the NYN/PIN family of ribonucleases and is highly conserved in the Firmicutes, the Cyanobacteria and the chloroplasts of photosynthetic algae and plants. We have proposed a model in which Rae1 enters the A-site of ribosomes that are paused following translation of certain sequences that are still ill-defined. In the only case identified thus far, Rae1 cleaves between a conserved glutamate and lysine codon during translation of a short peptide called S1025. Certain other codons are also tolerated on either side of the cleavage site, but these are recognized less efficiently. The model of Rae1 docked in the A-site allows us to make predictions about which conserved residues may be important for recognition of mRNA, the tRNA in the adjacent P-site and binding to the 50S ribosome subunit.

Nonconventional localizations of cytosolic aminoacyl-tRNA synthetases in yeast and human cells.

By definition, cytosolic aminoacyl-tRNA synthetases (aaRSs) should be restricted to the cytosol of eukaryotic cells where they supply translating ribosomes with their aminoacyl-tRNA substrates. However, it has been shown that other translationally-active compartments like mitochondria and plastids can simultaneously contain the cytosolic aaRS and its corresponding organellar ortholog suggesting that both forms do not share the same organellar function. In addition, a fair number of cytosolic aaRSs have also been found in the nucleus of cells from several species. Hence, these supposedly cytosolic-restricted enzymes have instead the potential to be multi-localized. As expected, in all examples that were studied so far, when the cytosolic aaRS is imported inside an organelle that already contains its bona fide corresponding organellar-restricted aaRSs, the cytosolic form was proven to exert a nonconventional and essential function. Some of these essential functions include regulating homeostasis and protecting against various stresses. It thus becomes critical to assess meticulously the subcellular localization of each of these cytosolic aaRSs to unravel their additional roles. With this objective in mind, we provide here a review on what is currently known about cytosolic aaRSs multi-compartmentalization and we describe all commonly used protocols and procedures for identifying the compartments in which cytosolic aaRSs relocalize in yeast and human cells.

The conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome.

Using a combination of biochemical, structural probing and rapid kinetics techniques we reveal for the first time that the universally conserved translational GTPase (trGTPase) HflX binds to the E-site of the 70S ribosome and that its GTPase activity is modulated by peptidyl transferase centre (PTC) and peptide exit tunnel (PET) binding antibiotics, suggesting a previously undescribed mode of action for these antibiotics. Our rapid kinetics studies reveal that HflX functions as a ribosome splitting factor that disassembles the 70S ribosomes into its subunits in a nucleotide dependent manner. Furthermore, our probing and hydrolysis studies show that the ribosome is able to activate trGTPases bound to its E-site. This is, to our knowledge, the first case in which the hydrolytic activity of a translational GTPase is not activated by the GTPase activating centre (GAC) in the ribosomal A-site. Furthermore, we provide evidence that the bound state of the PTC is able to regulate the GTPase activity of E-site bound HflX.

The ribosome uses two active mechanisms to unwind messenger RNA during translation.

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs.

Methylation of histone H3 lysine 9 occurs during translation.

Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9), a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed.

Ribosomal Peptide Syntheses from Activated Substrates Reveal Rate Limitation by an Unexpected Step at the Peptidyl Site.

Protein synthesis (translation) is central to cellular function and antibiotic development. Interestingly, the key chemical step of translation, peptide bond formation, is among the slower enzymatic reactions. The reason for this remains controversial because of reliance on studies using highly modified, severely minimized, or unreactive substrate analogues. Here, we investigated this problem by fast kinetics using full-length aminoacyl-tRNA substrates with atomic substitutions that activated the ester electrophile. While trifluoro substitution of hydrogens in nonconserved positions of the peptidyl-site substrate dramatically increased the ester reactivity in solution assays, a large hastening of the combined rates of ribosomal accommodation and peptidyl transfer was observed only with a slowly reacting aminoacyl-site nucleophile, proline. With a fast-reacting A-site nucleophile, phenylalanine, effects did not correlate at all with electrophilicities. As effects were observed using the same, natural, aminoacyl-tRNA at the A site and all rates of accommodation/peptidyl transfer were pH dependent, we concluded that rate limitation was not by A-site accommodation but rather by peptidyl transfer and a hitherto unexpected step at the P site. This new slow step, which we term P-site accommodation, has implications for the activation or inhibition of ribosome function in vitro and in vivo.