Aminoglycoside interactions and impacts on the eukaryotic ribosome

Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages (initiation, elongation, termination, and recycling) of the translation mechanism. The propensity of aminoglycosides to induce miscoding errors that suppress the termination of protein synthesis supports their potential as therapeutic interventions in human diseases associated with premature termination codons (PTCs). However, the sites of interaction of aminoglycosides with the eukaryotic ribosome and their modes of action in eukaryotic translation remain largely unexplored. Here, we use the combination of X-ray crystallography and single-molecule FRET analysis to reveal the interactions of distinct classes of aminoglycosides with the 80S eukaryotic ribosome. Crystal structures of the 80S ribosome in complex with paromomycin, geneticin (G418), gentamicin, and TC007, solved at 3.3- to 3.7-A resolution, reveal multiple aminoglycoside-binding sites within the large and small subunits, wherein the 6'-hydroxyl substituent in ring I serves as a key determinant of binding to the canonical eukaryotic ribosomal decoding center. Multivalent binding interactions with the human ribosome are also evidenced through their capacity to affect large-scale conformational dynamics within the pretranslocation complex that contribute to multiple aspects of the translation mechanism. The distinct impacts of the aminoglycosides examined suggest that their chemical composition and distinct modes of interaction with the ribosome influence PTC read-through efficiency. These findings provide structural and functional insights into aminoglycoside-induced impacts on the eukaryotic ribosome and implicate pleiotropic mechanisms of action beyond decoding

Bulk-solvent correction in large macromolecular structures.

International audienceThe estimation of the bulk-solvent contribution to the diffraction of a macromolecular crystal makes use of a solvent mask which delimits the bulk-solvent regions in the crystal. It is shown that the way this mask is usually defined in CNS contains a bias which can lead to absurd results in the case of very large structures, where the calculations can only be made on relatively coarse grids. A modified procedure is described and applied to 70S ribosome data at 5.5 A resolution. The B factor affecting the bulk solvent is also discussed. Even in this case of very high and widely variable atomic B factors, it seems sufficient to consider a constant and isotropic B factor for the bulk solvent. This is initially set to the average value of the atomic B factor, but can be refined

Structured mRNAs regulate translation initiation by binding to the platform of the ribosome.

International audienceGene expression can be regulated at the level of initiation of protein biosynthesis via structural elements present at the 5' untranslated region of mRNAs. These folded mRNA segments may bind to the ribosome, thus blocking translation until the mRNA unfolds. Here, we report a series of cryo-electron microscopy snapshots of ribosomal complexes directly visualizing either the mRNA structure blocked by repressor protein S15 or the unfolded, active mRNA. In the stalled state, the folded mRNA prevents the start codon from reaching the peptidyl-tRNA (P) site inside the ribosome. Upon repressor release, the mRNA unfolds and moves into the mRNA channel allowing translation initiation. A comparative structure and sequence analysis suggests the existence of a universal stand-by site on the ribosome (the 30S platform) dedicated for binding regulatory 5' mRNA elements. Different types of mRNA structures may be accommodated during translation preinitiation and regulate gene expression by transiently stalling the ribosome

Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding.

International audienceThe ribosome of Thermus thermophilus was cocrystallized with initiator transfer RNA (tRNA) and a structured messenger RNA (mRNA) carrying a translational operator. The path of the mRNA was defined at 5.5 angstroms resolution by comparing it with either the crystal structure of the same ribosomal complex lacking mRNA or with an unstructured mRNA. A precise ribosomal environment positions the operator stem-loop structure perpendicular to the surface of the ribosome on the platform of the 30S subunit. The binding of the operator and of the initiator tRNA occurs on the ribosome with an unoccupied tRNA exit site, which is expected for an initiation complex. The positioning of the regulatory domain of the operator relative to the ribosome elucidates the molecular mechanism by which the bound repressor switches off translation. Our data suggest a general way in which mRNA control elements must be placed on the ribosome to perform their regulatory task

Dimerization of long hibernation promoting factor from Staphylococcus aureus: Structural analysis and biochemical characterization

International audienceStaphylococcus aureus hibernation promoting factor (SaHPF) is responsible for the formation of 100S ribosome dimers, which in turn help this pathogen to reduce energy spent under unfavorable conditions. Ribosome dimer formation strongly depends on the dimerization of the C-terminal domain of SaHPF (CTDSaHPF). In this study, we solved the crystal structure of CTDSaHPF at 1.6 Å resolution and obtained a precise arrangement of the dimer interface. Residues Phe160, Val162, Thr171, Ile173, Tyr175, Ile185 andThr187 in the dimer interface of SaHPF protein were mutated and the effects were analyzed for the formation of 100S disomes of ribosomes isolated from S. aureus. It was shown that substitution of any of single residues Phe160, Val162, Ile173, Tyr175 and Ile185 in the SaHPF homodimer interface abolished the ribosome dimerization in vitro

Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome.

International audienceInitiation of protein synthesis is a universally conserved event that requires initiation factors IF1, IF2 and IF3 in prokaryotes. IF2 is a GTPase essential for binding initiator transfer RNA to the 30S ribosomal subunit and recruiting the 50S subunit into the 70S initiation complex. We present two cryo-EM structures of the assembled 70S initiation complex comprising mRNA, fMet-tRNA(fMet) and IF2 with either a non-hydrolyzable GTP analog or GDP. Transition from the GTP-bound to the GDP-bound state involves substantial conformational changes of IF2 and of the entire ribosome. In the GTP analog-bound state, IF2 interacts mostly with the 30S subunit and extends to the initiator tRNA in the peptidyl (P) site, whereas in the GDP-bound state IF2 steps back and adopts a 'ready-to-leave' conformation. Our data also provide insights into the molecular mechanism guiding release of IF1 and IF3