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In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. In eukaryotes, the role of eIF5B in ribosomal subunit joining is established and structural data showing eIF5B bound to the full ribosome were obtained. To achieve its function, eIF5B collaborates with eIF1A. However, structural data illustrating how these two factors interact on the small ribosomal subunit have long been awaited. The role of the archaeal counterparts, aIF5B and aIF1A, remains to be extensively addressed. Here, we study the late steps of Pyrococcus abyssi translation initiation. Using in vitro reconstituted initiation complexes and light scattering, we show that aIF5B bound to GTP accelerates subunit joining without the need for GTP hydrolysis. We report the crystallographic structures of aIF5B bound to GDP and GTP and analyze domain movements associated to these two nucleotide states. Finally, we present the cryo-EM structure of an initiation complex containing 30S bound to mRNA, Met-tRNAiMet, aIF5B and aIF1A at 2.7 Å resolution. Structural data shows how archaeal 5B and 1A factors cooperate to induce a conformation of the initiator tRNA favorable to subunit joining. Archaeal and eukaryotic features of late steps of translation initiation are discussed.

Aberrantly slow mRNA translation leads to ribosome stalling and subsequent collision with the trailing neighbor. Ribosome collisions have recently been shown to act as stress sensors in the cell, with the ability to trigger stress responses balancing survival and apoptotic cell-fate decisions depending on the stress level. However, we lack a molecular understanding of the reorganization of translation processes over time in mammalian cells exposed to an unresolved collision stress. Here we visualize the effect of a persistent collision stress on translation usingin situcryo electron tomography. We observe that low dose anisomycin collision stress leads to the stabilization of Z-site bound tRNA on elongating 80S ribosomes, as well as to the accumulation of an off-pathway 80S complex possibly resulting from collision splitting events. We visualize collided disomesin situ, occurring on compressed polysomes and revealing a stabilized geometry involving the Z-tRNA and L1 stalk on the stalled ribosome, and eEF2 bound to its collided rotated-2 neighbor. In addition, non-functional post-splitting 60S complexes accumulate in the stressed cells, indicating a limiting Ribosome associated Quality Control clearing rate. Finally, we observe the apparition of tRNA-bound aberrant 40S complexes shifting with the stress timepoint, suggesting a succession of different initiation inhibition mechanisms over time. Altogether, our work visualizes the changes of translation complexes under persistent collision stress in mammalian cells, indicating how perturbations in initiation, elongation and quality control processes contribute to an overall reduced protein synthesis.SummaryUsingin situcryo electron tomography we visualized the reorganization of mammalian translation processes during a persistent collision stress.

Amino acids (AAs) with a noncanonical backbone would be a valuable tool for protein engineering, enabling new structural motifs and building blocks. To incorporate them into an expanded genetic code, the first, key step is to obtain an appropriate aminoacyl-tRNA synthetase (aaRS). Currently, directed evolution is not available to optimize such AAs, since an appropriate selective pressure is not available. Computational protein design (CPD) is an alternative. We used a new CPD method to redesign MetRS and increase its activity towardsβ-Met, which has an extra backbone methylene. The new method considered a few active site positions for design and used a Monte Carlo exploration of the corresponding sequence space. During the exploration, a bias energy was adaptively learned, such that the free energy landscape of the apo enzyme was flattened. Enzyme variants could then be sampled, in the presence of the ligand and the bias energy, according to theirβ-Met binding affinities. Eleven predicted variants were chosen for experimental testing; all exhibited detectable activity forβ-Met adenylation. Top predicted hits were characterized experimentally in detail. Dissociation constants, catalytic rates, and Michaelis constants for bothα-Met andβ-Met were measured. The best mutant retained a preference forα-Met overβ-Met; however, the preference was reduced, compared to the wildtype, by a factor of 29. For this mutant, high resolution crystal structures were obtained in complex with bothα-Met andβ-Met, indicating that the predicted, active conformation ofβ-Met in the active site was retained.Author summaryAmino acids (AAs) with a noncanonical backbone would be valuable for protein engineering, enabling new structural motifs. To incorporate them into an expanded genetic code, the key step is to obtain an appropriate aminoacyl-tRNA synthetase (aaRS). Currently, directed evolution is not available to optimize such AAs. Computational protein design is an alternative. We used a new method to redesign MetRS and increase its activity towardsβ-Met, which has an extra backbone methylene. The method considered a few active site positions for design and used a Monte Carlo exploration of sequence space, during which a bias energy was adaptively learned, such that the free energy landscape of the apo enzyme was flattened. Enzyme variants could then be sampled, in the presence of the ligand and the bias energy, according to theirβ-Met binding affinities. Eleven predicted variants were chosen for experimental testing; all exhibited detectableβ-Met adenylation activity. Top hits were characterized experimentally in detail. The best mutant had its preference forα-Met overβ-Met reduced by a factor of 29. Crystal structures indicated that the predicted, active conformation ofβ-Met in the active site was retained.

Small-angle X-ray scattering (SAXS) has become an indispensable tool in structural biology, complementing atomic-resolution techniques. It is sensitive to the electron-density difference between solubilized biomacromolecules and the buffer, and provides information on molecular masses, particle dimensions and interactions, low-resolution conformations and pair distance-distribution functions. When SAXS data are recorded at multiple contrasts, i.e. at different solvent electron densities, it is possible to probe, in addition to their overall shape, the internal electron-density profile of biomacromolecular assemblies. Unfortunately, contrast-variation SAXS has been limited by the range of solvent electron densities attainable using conventional co-solutes (for example sugars, glycerol and salt) and by the fact that some biological systems are destabilized in their presence. Here, SAXS contrast data from an oligomeric protein and a protein–RNA complex are presented in the presence of iohexol and Gd-HPDO3A, two electron-rich molecules that are used in biomedical imaging and that belong to the families of iodinated and lanthanide-based complexes, respectively. Moderate concentrations of both molecules allowed solvent electron densities matching those of proteins to be attained. While iohexol yielded higher solvent electron densities (per mole), it interacted specifically with the oligomeric protein and precipitated the protein–RNA complex. Gd-HPDO3A, while less efficient (per mole), did not disrupt the structural integrity of either system, and atomic models could be compared with the SAXS data. Due to their elevated solubility and electron density, their chemical inertness, as well as the possibility of altering their physico-chemical properties, lanthanide-based complexes represent a class of molecules with promising potential for contrast-variation SAXS experiments on diverse biomacromolecular systems.

Recently, we observed the first example of a left‐handed G‐quadruplex structure formed by natural DNA, named Z‐G4 . We analysed the Z‐G4 structure and inspected its primary 28‐nt sequence in order to identify motifs that convey the unique left‐handed twist. Using circular dichroism spectroscopy, NMR spectroscopy, and X‐ray crystallography, we revealed a minimal sequence motif of 12 nt (GTGGTGGTGGTG) for formation of the left‐handed DNA G‐quadruplex, which is found to be highly abundant in the human genome. A systematic analysis of thymine loop mutations revealed a moderate sequence tolerance, which would further broaden the space of sequences prone to left‐handed G‐quadruplex formation. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Accepted version

G-quadruplex (G4) DNA structures with a left-handed backbone progression have unique and conserved structural features. Studies on sequence dependency of the structures revealed the prerequisites and some minimal motifs required for left-handed G4 formation. To extend the boundaries, we explore the adaptability of left-handed G4s towards the existence of bulges. Here we present two X-ray crystal structures and an NMR solution structure of left-handed G4s accommodating one, two and three bulges. Bulges in left-handed G4s show distinct characteristics as compared to those in right-handed G4s. The elucidation of intricate structural details will help in understanding the possible roles and limitations of these unique structures. Ministry of Education (MOE) Nanyang Technological University National Research Foundation (NRF) Published version Singapore National Research Foundation Investigatorship [NRF-NRFI2017-09]; Singapore Ministry of Education Academic Research Fund Tier 2 [MOE2015-T2- 1092]; Nanyang Technological University (NTU Singapore) grants (to A.T.P.). Funding for open access charge: Nanyang Technological University.

A recent study on the left-handed G-quadruplex (LHG4) DNA revealed a 12-nt minimal motif GTGGTGGTGGTG with the ability to independently form an LHG4 and to drive an adjacent sequence to LHG4 formation. Here we have identified a second LHG4-forming motif, GGTGGTGGTGTG, and determined the X-ray crystal structure of an LHG4 involving this motif. Our structural analysis indicated the role of split guanines and single thymine loops in promoting LHG4 formation. Ministry of Education (MOE) National Research Foundation (NRF) Accepted version NRF-NRFI2017-09

Archaeal translation initiation occurs within a macromolecular complex containing the small ribosomal subunit (30S) bound to mRNA, initiation factors aIF1, aIF1A and the ternary complex aIF2:GDPNP:Met-tRNAiMet. Here, we determine the cryo-EM structure of a 30S:mRNA:aIF1A:aIF2:GTP:Met-tRNAiMet complex from Pyrococcus abyssi at 3.2 Å resolution. It highlights archaeal features in ribosomal proteins and rRNA modifications. We find an aS21 protein, at the location of eS21 in eukaryotic ribosomes. Moreover, we identify an N-terminal extension of archaeal eL41 contacting the P site. We characterize 34 N4-acetylcytidines distributed throughout 16S rRNA, likely contributing to hyperthermostability. Without aIF1, the 30S head is stabilized and initiator tRNA is tightly bound to the P site. A network of interactions involving tRNA, mRNA, rRNA modified nucleotides and C-terminal tails of uS9, uS13 and uS19 is observed. Universal features and domain-specific idiosyncrasies of translation initiation are discussed in light of ribosomal structures from representatives of each domain of life., Coureux et al. describe a cryo-EM structure of a 30S initiation complex of Pyrococcus abyssi at 3.2Å resolution. The structure uncovers a novel archaeal ribosomal protein aS21, N-terminal extension of eL41 and brings insights into base modifications of the rRNA.

International audience; Translation initiation (TI) allows accurate selection of the initiation codon on a messenger RNA (mRNA) and defines the reading frame. In all domains of life, translation initiation generally occurs within a macromolecular complex made up of the small ribosomal subunit, the mRNA, a specialized methionylated initiator tRNA, and translation initiation factors (IFs). Once the start codon is selected at the P site of the ribosome and the large subunit is associated, the IFs are released and a ribosome competent for elongation is formed. However, even if the general principles are the same in the three domains of life, the molecular mechanisms are different in bacteria, eukaryotes, and archaea and may also vary depending on the mRNA. Because TI mechanisms have evolved lately, their studies bring important information about the evolutionary relationships between extant organisms. In this context, recent structural data on ribosomal complexes and genome-wide studies are particularly valuable. This review focuses on archaeal translation initiation highlighting its relationships with either the eukaryotic or the bacterial world. Eukaryotic features of the archaeal small ribosomal subunit are presented. Ribosome evolution and TI mechanisms diversity in archaeal branches are discussed. Next, the use of leaderless mRNAs and that of leadered mRNAs having Shine-Dalgarno sequences is analyzed. Finally, the current knowledge on TI mechanisms of SD-leadered and leaderless mRNAs is detailed.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design., Author summary Designed enzymes are of major interest. Experimental directed evolution still has significant limitations, and computational approaches are another route. Enzymes must be stable, bind substrates, and be powerful catalysts. It is challenging to design for all these properties. A method to design substrate binding was proposed recently. It used an adaptive Monte Carlo method to explore mutations of a few amino acids near the substrate. A bias energy was gradually “learned” such that, in the absence of the ligand, the simulation visited most of the possible protein mutations with comparable probabilities. Remarkably, a simulation of the protein:ligand complex, including the bias, will then preferentially sample tight-binding sequences. We generalized the method to design binding specificity. We tested it for the methionyl-tRNA synthetase enzyme, which has been engineered in order to expand the genetic code. We redesigned the enzyme to obtain variants with low activation free energies for the catalytic step. The variants proposed by the simulations were shown experimentally to be active, and the predicted activation free energies were in reasonable agreement with the experimental values. We expect the new method will become the paradigm for computational enzyme design.

International audience; Polypeptides containing β-amino acids are attractive tools for the design of novel proteins having unique properties of medical or industrial interest. Incorporation of β-amino acids in vivo requires the development of efficient aminoacyl-tRNA synthetases specific of these non-canonical amino acids. Here, we have performed a detailed structural and biochemical study of the recognition and use of β3-Met by Escherichia coli methionyl-tRNA synthetase (MetRS). We show that MetRS binds β3-Met with a 24-fold lower affinity but catalyzes the esterification of the non-canonical amino acid onto tRNA with a rate lowered by three orders of magnitude. Accurate measurements of the catalytic parameters required careful consideration of the presence of contaminating α-Met in β3-Met commercial samples. The 1.45 Å crystal structure of the MetRS: β3-Met complex shows that β3-Met binds the enzyme essentially like α-Met, but the carboxylate moiety is mobile and not adequately positioned to react with ATP for aminoacyl adenylate formation. This study provides structural and biochemical bases for engineering MetRS with improved β3-Met aminoacylation capabilities.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are complementary. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We extend the method to the design of an enzyme for specific transition state binding, i.e., for catalytic power. We consider methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. MetRS and other synthetases have been extensively redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. We redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered, and mutants predicted to bind MetAMP were characterized experimentally and found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We expect the present method will become the paradigm for computational enzyme design.

Polypeptides containing β-amino acids are attractive tools for the design of novel proteins having unique properties of medical or industrial interest. Incorporation of β-amino acids in vivo requires the development of efficient aminoacyl-tRNA synthetases specific of these non-canonical amino acids. Here, we have performed a detailed structural and biochemical study of the recognition and use of β

Summary: cGMP is critical to a variety of cellular processes, but the available tools to interfere with endogenous cGMP lack cellular and subcellular specificity. We introduce SponGee, a genetically encoded chelator of this cyclic nucleotide that enables in vitro and in vivo manipulations in single cells and in biochemically defined subcellular compartments. SponGee buffers physiological changes in cGMP concentration in various model systems while not affecting cAMP signals. We provide proof-of-concept strategies by using this tool to highlight the role of cGMP signaling in vivo and in discrete subcellular domains. SponGee enables the investigation of local cGMP signals in vivo and paves the way for therapeutic strategies that prevent downstream signaling activation. : Ros et al. developed SponGee, a genetically encoded cGMP chelator that enables the manipulation of this second messenger in single cells with subcellular specificity. SponGee alters the migration of developing cortical neurons in vivo. Lipid raft targeting of SponGee prevents axon repulsion, in contrast to exclusion from this subcellular compartment. Keywords: cGMP buffer, subcellular compartment, single cell pharmacology, axon guidance, neuronal migration, lipid grafts, genetically encoded, FRET, ThPDE5VV, PKG

Analogous to the B- and Z-DNA structures in double-helix DNA, there exist both right- and left-handed quadruple-helix (G-quadruplex) DNA. Numerous conformations of right-handed and a few left-handed G-quadruplexes were previously observed, yet they were always identified separately. Here, we present the NMR solution and X-ray crystal structures of a right- and left-handed hybrid G-quadruplex. The structure reveals a stacking interaction between two G-quadruplex blocks with different helical orientations and displays features of both right- and left-handed G-quadruplexes. An analysis of loop mutations suggests that single-nucleotide loops are preferred or even required for the left-handed G-quadruplex formation. The discovery of a right- and left-handed hybrid G-quadruplex further expands the polymorphism of G-quadruplexes and is potentially useful in designing a left-to-right junction in G-quadruplex engineering. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Published version

International audience; The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocy-tosed receptors, such as a5b1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled-coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyos-telium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients.

International audience; Eukaryotic and archaeal translation initiation processes involve a heterotrimeric GTPase e/aIF2 crucial for accuracy of start codon selection. In eu-karyotes, the GTPase activity of eIF2 is assisted by a GTPase-activating protein (GAP), eIF5. In ar-chaea, orthologs of eIF5 are not found and aIF2 GT-Pase activity is thought to be non-assisted. However , no in vitro GTPase activity of the archaeal factor has been reported to date. Here, we show that aIF2 significantly hydrolyses GTP in vitro. Within aIF2␥, H97, corresponding to the catalytic histidine found in other translational GTPases, and D19, from the GKT loop, both participate in this activity. Several high-resolution crystal structures were determined to get insight into GTP hydrolysis by aIF2␥. In particular, a crystal structure of the H97A mutant was obtained in the presence of non-hydrolyzed GTP. This structure reveals the presence of a second magnesium ion bound to GTP and D19. Quantum chemical/molecular mechanical simulations support the idea that the second magnesium ion may assist GTP hydrolysis by helping to neutralize the developing negative charge in the transition state. These results are discussed in light of the absence of an identified GAP in archaea to assist GTP hydrolysis on aIF2.

Translation initiation in eukaryotes and archaea involves a methionylated initiator tRNA delivered to the ribosome in a ternary complex with e/aIF2 and GTP. Eukaryotic and archaeal initiator tRNAs contain a highly conserved A1–U72 base pair at the top of the acceptor stem. The importance of this base pair to discriminate initiator tRNAs from elongator tRNAs has been established previously using genetics and biochemistry. However, no structural data illustrating how the A1–U72 base pair participates in the accurate selection of the initiator tRNAs by the translation initiation systems are available. Here, we describe the crystal structure of a mutant E. coli initiator tRNAfMetA1–U72, aminoacylated with methionine, in which the C1:A72 mismatch at the end of the tRNA acceptor stem has been changed to an A1–U72 base pair. Sequence alignments show that the mutant E. coli tRNA is a good mimic of archaeal initiator tRNAs. The crystal structure, determined at 2.8 Å resolution, shows that the A1–U72 pair adopts an unusual arrangement. A1 is in a syn conformation and forms a single H-bond interaction with U72. This interaction requires protonation of the N1 atom of A1. Moreover, the 5′ phosphoryl group folds back into the major groove of the acceptor stem and interacts with the N7 atom of G2. A possible role of this unusual geometry of the A1–U72 pair in the recognition of the initiator tRNA by its partners during eukaryotic and archaeal translation initiation is discussed.

Eukaryotic and archaeal translation initiation complexes have a common structural core comprising e/aIF1, e/aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNAiMet) and mRNA bound to the small ribosomal subunit. e/aIF2 plays a crucial role in this process but how this factor controls start codon selection remains unclear. Here, we present cryo-EM structures of the full archaeal 30S initiation complex showing two conformational states of the TC. In the first state, the TC is bound to the ribosome in a relaxed conformation with the tRNA oriented out of the P site. In the second state, the tRNA is accommodated within the peptidyl (P) site and the TC becomes constrained. This constraint is compensated by codon/anticodon base pairing, whereas in the absence of a start codon, aIF2 contributes to swing out the tRNA. This spring force concept highlights a mechanism of codon/anticodon probing by the initiator tRNA directly assisted by aIF2., Initiation factor eIF2, common to eukaryotes and archaea, is a central actor in translation initiation. Here the authors describe two cryo-EM structures of archaeal 30S initiation complexes that provide a novel view of the central role that e/aIF2 plays in start codon selection.

International audience; Heterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2-GDPNP-tRNA complex. © The Author(s) 2012. Published by Oxford University Press.

Translation initiation in eukaryotes and archaea involves a methionylated initiator tRNA delivered to the ribosome in a ternary complex with e/aIF2 and GTP. Eukaryotic and archaeal initiator tRNAs contain a highly conserved A1–U72 base pair at the top of the acceptor stem. The importance of this base pair to discriminate initiator tRNAs from elongator tRNAs has been established previously using genetics and biochemistry. However, no structural data illustrating how the A1–U72 base pair participates in the accurate selection of the initiator tRNAs by the translation initiation systems are available. Here, we describe the crystal structure of a mutant E. coli initiator tRNAfMetA1–U72, aminoacylated with methionine, in which the C1:A72 mismatch at the end of the tRNA acceptor stem has been changed to an A1–U72 base pair. Sequence alignments show that the mutant E. coli tRNA is a good mimic of archaeal initiator tRNAs. The crystal structure, determined at 2.8 Å resolution, shows that the A1–U72 pair adopts an unusual arrangement. A1 is in a syn conformation and forms a single H-bond interaction with U72. This interaction requires protonation of the N1 atom of A1. Moreover, the 5′ phosphoryl group folds back into the major groove of the acceptor stem and interacts with the N7 atom of G2. A possible role of this unusual geometry of the A1–U72 pair in the recognition of the initiator tRNA by its partners during eukaryotic and archaeal translation initiation is discussed.

International audience; In all organisms, translational initiation takes place on the small ribosomal subunit and two classes of methionine tRNA are present. The initiator is used exclusively for initiation of protein synthesis while the elongator is used for inserting methionine internally in the nascent polypeptide chain. The crystal structure of Escherichia coli initiator tRNA(f)(Met) has been solved at 3.1 A resolution. The anticodon region is well-defined and reveals a unique structure, which has not been described in any other tRNA. It encompasses a Cm32*A38 base pair with a peculiar geometry extending the anticodon helix, a base triple between A37 and the G29-C41 pair in the major groove of the anticodon stem and a modified stacking organization of the anticodon loop. This conformation is associated with the three GC basepairs in the anticodon stem, characteristic of initiator tRNAs and suggests a mechanism by which the translation initiation machinery could discriminate the initiator tRNA from all other tRNAs.

Initiation of translation in eukaryotes and in archaea involves eukaryotic/archaeal initiation factor (e/aIF)1 and the heterotrimeric initiation factor e/aIF2. In its GTP-bound form, e/aIF2 provides the initiation complex with Met–tRNA i Met . After recognition of the start codon by initiator tRNA, e/aIF1 leaves the complex. Finally, e/aIF2, now in a GDP-bound form, loses affinity for Met–tRNA i Met and dissociates from the ribosome. Here, we report a 3D structure of an aIF2 heterotrimer from the archeon Sulfolobus solfataricus obtained in the presence of GDP. Our report highlights how the two-switch regions involved in formation of the tRNA-binding site on subunit γ exchange conformational information with α and β. The zinc-binding domain of β lies close to the guanine nucleotide and directly contacts the switch 1 region. As a result, switch 1 adopts a not yet described conformation. Moreover, unexpectedly for a GDP-bound state, switch 2 has the “ON” conformation. The stability of these conformations is accounted for by a ligand, most probably a phosphate ion, bound near the nucleotide binding site. The structure suggests that this GDP–inorganic phosphate (Pi) bound state of aIF2 may be proficient for tRNA binding. Recently, it has been proposed that dissociation of eIF2 from the initiation complex is closely coupled to that of Pi from eIF2γ upon start codon recognition. The nucleotide state of aIF2 shown here is indicative of a similar mechanism in archaea. Finally, we consider the possibility that release of Pi takes place after e/aIF2γ has been informed of e/aIF1 dissociation by e/aIF2β.

International audience; Eukaryotic initiation factor 2 (eIF2), a heterotrimeric guanosine triphosphatase, has a central role in protein biosynthesis by supplying methionylated initiator tRNA to the ribosomal translation initiation complex and by serving as a target for translational control in response to stress. Recent work identified a novel step indispensable for eIF2 function: assembly of eIF2 from its three subunits by the cell proliferation protein Cdc123. We report the first crystal structure of a Cdc123 representative, that from Schizosaccharomyces pombe, both isolated and bound to domain III of Saccharomyces cerevisiae eIF2 gamma. The structures show that Cdc123 resembles enzymes of the ATP-grasp family. Indeed, Cdc123 binds ATP-Mg2+, and conserved residues contacting ATP-Mg2+ are essential for Cdc123 to support eIF2 assembly and cell viability. A docking of eIF2 alpha gamma onto Cdc123, combined with genetic and biochemical experiments, allows us to propose a model explaining how Cdc123 participates in the biogenesis of eIF2 through facilitating assembly of eIF2 gamma to eIF2 alpha.

Significance DNA can adopt diverse structural conformations including duplexes, triplexes, and quadruplexes. Four-stranded structures known as G-quadruplexes have been implicated in cellular processes and found potential applications in therapeutics and nanotechnology. G-quadruplex structures are highly polymorphic, but so far, only right-handed helical forms have been observed. Here we present the NMR solution and X-ray crystal structures of an unprecedented left-handed DNA G-quadruplex, containing structural features that can be exploited as unique recognition elements.

Summary Eukaryotic and archaeal initiation factors 2 (e/aIF2) are heterotrimeric proteins (αβγ) supplying the small subunit of the ribosome with methionylated initiator tRNA. This study reports the crystallographic structure of an aIF2αγ heterodimer from Sulfolobus solfataricus bound to Gpp(NH)p-Mg 2+ . aIF2γ is in a closed conformation with the G domain packed on domains II and III. The C-terminal domain of aIF2α interacts with domain II of aIF2γ. Conformations of the two switch regions involved in GTP binding are similar to those encountered in an EF1A:GTP:Phe-tRNA Phe complex. Comparison with the EF1A structure suggests that only the γ subunit of the aIF2αγ heterodimer contacts tRNA. Because the α subunit markedly reinforces the affinity of tRNA for the γ subunit, a contribution of the α subunit to the switch movements observed in the γ structure is considered.

International audience; Eukaryotic and archaeal initiation factor 2 (e- and aIF2, respectively) are heterotrimeric proteins (alphabetagamma) supplying the small subunit of the ribosome with methionylated initiator tRNA. The gamma subunit forms the core of the heterotrimer. It resembles elongation factor EF1-A and ensures interaction with Met-tRNA(i)(Met). In the presence of the alpha subunit, which is composed of three domains, the gamma subunit expresses full tRNA binding capacity. This study reports the crystallographic structure of the intact aIF2alpha subunit from the archaeon Pyrococcus abyssi and that of a derived C-terminal fragment containing domains 2 and 3. The obtained structures are compared with those of N-terminal domains 1 and 2 of yeast and human eIF2alpha and with the recently determined NMR structure of human eIF2alpha. We show that the three-domain organization in the alpha subunit is conserved in archaea and eukarya. Domains 1 and 2 form a rigid body linked to a mobile third domain. Sequence comparisons establish that the most conserved regions in the aIF2alpha polypeptide lie at opposite sides of the protein, within domain 1 and domain 3, respectively. These two domains are known to exhibit RNA binding capacities. We propose that domain 3, which is known to glue the alpha subunit onto the gamma subunit, participates in Met-tRNA(i)(Met) binding while domain 1 recognizes either rRNA or mRNA on the ribosome. Thereby, the observed structural mobility within the e- and aIF2alpha molecules would be an integral part of the biological function of this subunit in the heterotrimeric e- and aIF2alphabetagamma factors.

International audience; The 3-D structure of the peptidyl-tRNA hydrolase from the archaea Sulfolobus solfataricus has been solved at 1.8 A resolution. Homologues of this enzyme are found in archaea and eucarya. Bacteria display a different type of peptidyl-tRNA hydrolase that is also encountered in eucarya. In solution, the S. solfataricus hydrolase behaves as a dimer. In agreement, the crystalline structure of this enzyme indicates the formation of a dimer. Each protomer is made of a mixed five-stranded beta-sheet surrounded by two groups of two alpha-helices. The dimer interface is mainly formed by van der Waals interactions between hydrophobic residues belonging to the two N-terminal alpha1 helices contributed by two protomers. Site-directed mutagenesis experiments were designed for probing the basis of specificity of the archaeal hydrolase. Among the strictly conserved residues within the archaeal/eucaryal peptidyl-tRNA hydrolase family, three residues, K18, D86, and T90, appear of utmost importance for activity. They are located in the N-part of alpha1 and in the beta3-beta4 loop. K18 and D86, which form a salt bridge, might play a role in the catalysis thanks to their acid and basic functions, whereas the OH group of T90 could act as a nucleophile. These observations clearly distinguish the active site of the archaeal/eucaryal hydrolases from that of the bacterial/eucaryal ones, where a histidine is believed to serve as the catalytic base.

International audience; Initiation of protein synthesis in bacteria, mitochondria, and chloroplasts involves a formylated methionyl-tRNA species. Formylation of this tRNA is catalyzed by a methionyl-tRNA(f)(Met) formyltransferase (formylase). Upon inactivation of the gene encoding formylase, the growth rate of Escherichia coli is severely decreased. This behavior underlines the importance of formylation to give tRNA(Met) an initiator identity. Surprisingly, however, recent data [Li, Y., Holmes, W. B., Appling, D. R., and RajBhandary, U. L. (2000) J. Bacteriol. 182, 2886-2892] showed that the respiratory growth of Saccharomyces cerevisiaewas not sensitive to deprivation of the mitochondrial formylase. In the present study, we report conditions of temperature or of growth medium composition in which inactivation of the formylase gene indeed impairs the growth of a S. cerevisiae haploid strain. Therefore, some selective advantage can eventually be associated to the existence of a formylating activity in the fungal mitochondrion under severe growth conditions. Finally, the specificity toward tRNA of S. cerevisiae mitochondrial formylase was studied using E. coli initiator tRNA and mutants derived from it. Like its bacterial counterpart, this formylase recognizes nucleotidic features in the acceptor stem of mitochondrial initiator tRNA. This behavior markedly distinguishes the mitochondrial formylase of yeast from that of animals. Indeed, it was shown that bovine mitochondrial formylase mainly recognizes the side chain of the esterified methionine plus a purine-pyrimidine base pair in the D-stem of tRNA [Takeuchi, N., Vial, L., Panvert, M., Schmitt, E., Watanabe, K., Mechulam, Y., and Blanquet, S. (2001) J. Biol. Chem. 276, 20064-20068]. Distinct tRNA recognition mechanisms adopted by the formylases of prokaryotic, fungal, or mammalian origins are likely to reflect coevolution of these enzymes with their tRNA substrate. Each mechanism appears well suited to an efficient selection of the substrate within the pool of all tRNAs.

International audience; The heterotrimeric factor e/aIF2 plays a central role in eukaryotic/archaeal initiation of translation. By delivering the initiator methionyl-tRNA to the ribosome, e/aIF2 ensures specificity of initiation codon selection. The three subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus abyssi could be overproduced in Escherichia coli. The beta and gamma subunits each contain a tightly bound zinc. The large gamma subunit is shown to form the structural core for trimer assembly. The crystal structures of aIF2gamma, free or complexed to GDP-Mg(2+) or GDPNP-Mg(2+), were resolved at resolutions better than 2 A. aIF2gamma displays marked similarities to elongation factors. A distinctive feature of e/aIF2gamma is a subdomain containing a zinc-binding knuckle. Examination of the nucleotide-complexed aIF2gamma structures suggests mechanisms of action and tRNA binding properties similar to those of an elongation factor. Implications for the mechanism of translation initiation in both eukarya and archaea are discussed. In particular, positioning of the initiator tRNA in the ribosomal A site during the search for the initiation codon is envisaged.

International audience; Cyclodipeptide synthases form cyclodipeptides from two aminoacyl transfer RNAs. They use a ping-pong mechanism that begins with transfer of the aminoacyl moiety of the first aminoacyl tRNA onto a conserved serine, yielding an aminoacyl enzyme. Combining X-ray crystallography, site-directed mutagenesis and affinity labelling of the cyclodipeptide synthase AlbC, we demonstrate that the covalent intermediate reacts with the aminoacyl moiety of the second aminoacyl tRNA, forming a dipeptidyl enzyme, and identify the aminoacyl-binding sites of the aminoacyl tRNAs.

Cell growth inhibition by severald-amino acids can be explained by an in vivo production of d-aminoacyl-tRNA molecules.Escherichia coli and yeast cells express an enzyme,d-Tyr-tRNATyr deacylase, capable of recycling such d-aminoacyl-tRNA molecules into free tRNA andd-amino acid. Accordingly, upon inactivation of the genes of the above deacylases, the toxicity of d-amino acids increases. Orthologs of the deacylase are found in many cells. In this study, the crystallographic structure of dimeric E. coli d-Tyr-tRNATyr deacylase at 1.55 A resolution is reported. The structure corresponds to a β-barrel closed on one side by a β-sheet lid. This barrel results from the assembly of the two subunits. Analysis of the structure in relation with sequence homologies in the orthologous family suggests the location of the active sites at the carboxy end of the β-strands. The solved structure markedly differs from those of all other documented tRNA-dependent hydrolases.

Eubacterial peptidyl-tRNA hydrolase (PTH) recycles all N-blocked aminoacyl-tRNA molecules but initiator formyl-methionyl-tRNAfMet, the acceptor helix of which is characterized by a 1-72 mismatch. Positive selection by PTH of noninitiator tRNA molecules with a full 1-72 base pair is abolished, however, upon the removal of the 5'-phosphate. The tRNA 5'-phosphate plays therefore the role of a relay between the enzyme and the status of the 1-72 base pair. In this study, the receptor site for the 5'-phosphate of elongator peptidyl-tRNAs and the position at the surface of PTH of the 3'-end of complexed peptidyl-tRNA are identified by site-directed mutagenesis experiments. The former site comprehends two cationic side chains (K105 and R133) which are likely to clamp the phosphate. The second corresponds to a four asparagine cluster (N10, N21, N68, and N114). By using these two positional constraints, the acceptor arm of elongation factor Tu-bound Phe-tRNAPhe could be docked to PTH. Contacts involve the acceptor and TPsiC stems. By comparing the obtained 3D model to that of EF-Tu:Phe-tRNAPhe crystalline complex in which the 5'-phosphate of the ligand also lies between a K and an R side chain, we propose that, in both systems, the capacity of the 5'-phosphate of a tRNA to reach or not a receptor site is the main identity element governing generic selection of elongator tRNAs. On the other hand, while the 1-72 mismatch acts as an antideterminant for PTH or EF-Tu recognition, it behaves as a positive determinant for the formylation of initiator Met-tRNAfMet.

Formylation of the methionyl moiety esterified to the 3' end of tRNA(f)Met is a key step in the targeting of initiator tRNA towards the translation start machinery in prokaryotes. Accordingly, the presence of methionyl-tRNA(f)Met formyltransferase (FMT), the enzyme responsible for this formylation, is necessary for the normal growth of Escherichia coli. The present work describes the structure of crystalline E.coli FMT at 2.0 A, resolution. The protein has an N-terminal domain containing a Rossmann fold. This domain closely resembles that of the glycinamide ribonucleotide formyltransferase (GARF), an enzyme which, like FMT, uses N-10 formyltetrahydrofolate as formyl donor. However, FMT can be distinguished from GARF by a flexible loop inserted within its Rossmann fold. In addition, FMT possesses a C-terminal domain with a beta-barrel reminiscent of an OB fold. This latter domain provides a positively charged side oriented towards the active site. Biochemical evidence is presented for the involvement of these two idiosyncratic regions (the flexible loop in the N-terminal domain, and the C-terminal domain) in the binding of the tRNA substrate.

A few aminoacyl-tRNA synthetases are characterized by their ability to tightly bind a zinc atom. In the case of Escherichia coli methionyl-tRNA synthetase, a peptide of 21 residues (138--163) having a stable 3-D structure in solution is responsible for zinc binding [Fourmy, D., Meinnel, T., Mechulam, Y., & Blanquet, S. (1993) J. Mol. Biol. 231, 1066--1077; Fourmy, D., Dardel, F., & Blanquet, S. (1993) J. Mol. Biol. 231, 1078--1089]. This peptide, which belongs to a region connecting the two halves of the nucleotide-binding domain of methionyl-tRNA synthetase, is likely to form a modular domain close to the active center of the enzyme. In this study, two residues of the zinc-binding module, Asp138 and Arg139, are shown to contribute to the stabilization of the transition state of the reaction leading to the activation of methionine. Moreover, another residue, Phe135, located at the surface of the zinc-binding domain, is found to possibly guide the tRNA acceptor stem toward the active site of the enzyme during catalysis. The available data indicate an important functional role for the zinc-binding module of methionyl-tRNA synthetase, as well as for other modules connecting conserved secondary structure elements in the aminoacyl-tRNA synthetase family. The relation between the occurrence of such variable peptide modules and the expression of both substrate specificity and catalytic efficiency is discussed.

Methionyl-tRNA synthetase belongs to the class I aminoacyl-tRNA synthetase family characterized both by a catalytic center built around a Rossmann Fold and by the presence of the two peptidic marker sequences HIGH and KMSKS. In this study, the role of the 21HLGH24 motif of Escherichia coli methionyl-tRNA synthetase was studied in a systematic fashion by site-directed mutagenesis. It is shown that the two histidine residues play a crucial role in the catalysis of the methionyl adenylate formation by participating in the stabilisation of the ATP phosphate chain during the transition state. Moreover, the results suggest the involvement of the epsilon-imino group of histidine 21 and of the delta-imino group of histidine 24. Notably, the substitution of either the leucine or the glycine residue of the HLGH motif by alanine had no effect on the catalysis. From the data and from other studies with class I aminoacyl-tRNA synthetases, concomitant positive contributions of the HIGH and KMSKS sequences to reach the transition state of aminoacyl adenylate formation can be envisaged.

International audience; Eukaryotic and archaeal translation initiation factor 2 (e/aIF2) is a heterotrimeric GTPase that has a crucial role in the selection of the correct start codon on messenger RNA. We report the 5-Å resolution crystal structure of the ternary complex formed by archaeal aIF2 from Sulfolobus solfataricus, the GTP analog GDPNP and methionylated initiator tRNA. The 3D model is further supported by solution studies using small-angle X-ray scattering. The tRNA is bound by the α and γ subunits of aIF2. Contacts involve the elbow of the tRNA and the minor groove of the acceptor stem, but not the T-stem minor groove. We conclude that despite considerable structural homology between the core γ subunit of aIF2 and the elongation factor EF1A, these two G proteins of the translation apparatus use very different tRNA-binding strategies.

Selection of correct start codons on messenger RNAs is a key step required for faithful translation of the genetic message. Such a selection occurs in a complex process, during which a translation-competent ribosome assembles, eventually having in its P site a specialized methionyl-tRNA Met base-paired with the start codon on the mRNA. This chapter summarizes recent advances describing at the molecular level the successive steps involved in the process. Special emphasis is put on the roles of the three initiation factors and of the initiator tRNA, which are crucial for the efficiency and the specificity of the process. In particular, structural analyses concerning complexes containing ribosomal subunits, as well as detailed kinetic studies, have shed new light on the sequence of events leading to faithful initiation of protein synthesis in Bacteria

Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faeciummethyltransferase, as well as by the previously characterized aac(6′)-Ii that encodes a 6′-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m5C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m5C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 Å resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m5C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes' respective specificities for nucleotides C1404 and C1407.

Escherichia coli methionyl-tRNA synthetase recognizes its cognate tRNAs according to the sequence of the CAU anticodon. In order to identify residues of methionyl-tRNA synthetase involved in tRNA anticodon recognition, enzyme variants created by cassette mutagenesis were genetically screened for their acquired ability to charge tRNA(mMet) derivatives with an ochre or an amber anticodon and, consequently, to cause the suppression of a stop codon in an indicator gene. The selected enzymes are called suppressors. Mutations were firstly directed towards the region of the synthetase encompassing residues 451 to 467. Several dozens of suppressor enzymes were compared. Statistical analysis of the mutations suggested that the substitution of an Asp side-chain at position 456 was sufficient to render possible the charging of the ochre or amber suppressor tRNAs. Point mutants at this position were therefore constructed. Their behaviour demonstrated that various tRNA(Met) derivatives having a non-Met anticodon could be aminoacylated in vitro provided only that the side-chain of residue 456 was no longer acidic. In turn, the Asp456 residue is not essential to the CAU anticodon recognition, since its substitution does not impair the aminoacylation of wild-type tRNA(Met). The analysis was enlarged to a second region from residue 437 to residue 454. The mutagenesis highlighted two other positions, one of which, Asn452, appeared involved in wild-type tRNA(Met) binding. The second position, Asp449, plays a role very similar to that of Asp456. It is concluded that both Asp449 and 456 behave as "antideterminants", contributing together to the rejection by the enzyme of tRNAs carrying non-Met anticodons. Finally, it is shown that the activities of some particular methionyl-tRNA synthetase variants, which have been made indifferent to the sequence of the anticodon of a tRNA(Met), are tightly dependent on the presence of the nucleotide determinants specific to the acceptor stem of tRNA(Met).

In bacteria, the free amino group of the methionylated initiator tRNA is specifically modified by the addition of a formyl group. The functional relevance of such a formylation for the initiation of translation is not yet precisely understood. Advantage was taken here of the availability of the fmt gene, encoding the Escherichia coli Met-tRNA(fMet) formyltransferase, to measure the influence of variations in the level of formyltransferase activity on the involvement of various mutant tRNA(fMet) and tRNA(mMet) species in either initiation or elongation in vivo. The data obtained established that formylation plays a dual role, firstly, by dictating tRNA(fMet) to engage in the initiation of translation, and secondly, by preventing the misappropriation of this tRNA by the elongation apparatus. The importance of formylation in the initiator identity of tRNA(fMet) was further shown by the demonstration that elongator tRNA(fMet) may be used in initiation and no longer in elongation, provided that it is mutated into a formylatable species and is given the three G.C base pairs characteristic of the anticodon stem of initiator tRNAs.