Paola Fucini

Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites

The elongation cycle of protein synthesis involves the delivery of aminoacyl-transfer RNAs to the aminoacyl-tRNA-binding site (A site) of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A site. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner. Despite the availability of structures of various EF-G–ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryoelectron microscopy analysis to resolve two previously unseen subpopulations within Thermus thermophilus EF-G–ribosome complexes at subnanometre resolution, one of them with a partly translocated tRNA. Comparison of these substates reveals that translocation of tRNA on the 30S subunit parallels the swivelling of the 30S head and is coupled to unratcheting of the 30S body. Because the tRNA maintains contact with the peptidyl-tRNA-binding site (P site) on the 30S head and simultaneously establishes interaction with the exit site (E site) on the 30S platform, a novel intra-subunit ‘pe/E’ hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swivelled 30S-head conformation. These findings provide direct structural and mechanistic insight into the ‘missing link’ in terms of tRNA intermediates involved in the universally conserved translocation process.

The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning.

The oxazolidinones represent the first new class of antibiotics to enter into clinical usage within the past 30 years, but their binding site and mechanism of action has not been fully characterized. We have determined the crystal structure of the oxazolidinone linezolid bound to the Deinococcus radiodurans 50S ribosomal subunit. Linezolid binds in the A site pocket at the peptidyltransferase center of the ribosome overlapping the aminoacyl moiety of an A-site bound tRNA as well as many clinically important antibiotics. Binding of linezolid stabilizes a distinct conformation of the universally conserved 23S rRNA nucleotide U2585 that would be nonproductive for peptide bond formation. In conjunction with available biochemical data, we present a model whereby oxazolidinones impart their inhibitory effect by perturbing the correct positioning of tRNAs on the ribosome.

Inhibition of peptide bond formation by pleuromutilins: the structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin

Tiamulin, a prominent member of the pleuromutilin class of antibiotics, is a potent inhibitor of protein synthesis in bacteria. Up to now the effect of pleuromutilins on the ribosome has not been determined on a molecular level. The 3.5 Å structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin provides for the first time a detailed picture of its interactions with the 23S rRNA, thus explaining the molecular mechanism of the antimicrobial activity of the pleuromutilin class of antibiotics. Our results show that tiamulin is located within the peptidyl transferase center (PTC) of the 50S ribosomal subunit with its tricyclic mutilin core positioned in a tight pocket at the A‐tRNA binding site. Also, the extension, which protrudes from its mutilin core, partially overlaps with the P‐tRNA binding site. Thereby, tiamulin directly inhibits peptide bond formation. Comparison of the tiamulin binding site with other PTC targeting drugs, like chloramphenicol, clindamycin and streptogramins, may facilitate the design of modified or hybridized drugs that extend the applicability of this class of antibiotics.

Structural Insight into the Antibiotic Action of Telithromycin against Resistant Mutants

The crystal structure of the ketolide telithromycin bound to the Deinococcus radiodurans large ribosomal subunit shows that telithromycin blocks the ribosomal exit tunnel and interacts with domains II and V of the 23S RNA. Comparisons to other clinically relevant macrolides provided structural insights into its enhanced activity against macrolide-resistant strains.

Dissecting the ribosomal inhibition mechanisms of edeine and pactamycin: the universally conserved residues G693 and C795 regulate P-site RNA binding.

The crystal structures of the universal translation-initiation inhibitors edeine and pactamycin bound to ribosomal 30S subunit have revealed that edeine induces base pairing of G693:C795, residues that constitute the pactamycin binding site. Here, we show that base pair formation by addition of edeine inhibits tRNA binding to the P site by preventing codon-anticodon interaction and that addition of pactamycin, which rebreaks the base pair, can relieve this inhibition. In addition, edeine induces translational misreading in the A site, at levels comparable to those induced by the classic misreading antibiotic streptomycin. Binding of pactamycin between residues G693 and C795 strongly inhibits translocation with a surprising tRNA specificity but has no effect on translation initiation, suggesting that reclassification of this antibiotic is necessary. Collectively, these results suggest that the universally conserved G693:C795 residues regulate tRNA binding at the P site of the ribosome and influence translocation efficiency.

Alterations at the peptidyl transferase centre of the ribosome induced by the synergistic action of the streptogramins dalfopristin and quinupristin

BackgroundThe bacterial ribosome is a primary target of several classes of antibiotics. Investigation of the structure of the ribosomal subunits in complex with different antibiotics can reveal the mode of inhibition of ribosomal protein synthesis. Analysis of the interactions between antibiotics and the ribosome permits investigation of the specific effect of modifications leading to antimicrobial resistances.Streptogramins are unique among the ribosome-targeting antibiotics because they consist of two components, streptogramins A and B, which act synergistically. Each compound alone exhibits a weak bacteriostatic activity, whereas the combination can act bactericidal. The streptogramins A display a prolonged activity that even persists after removal of the drug. However, the mode of activity of the streptogramins has not yet been fully elucidated, despite a plethora of biochemical and structural data.ResultsThe investigation of the crystal structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with the clinically relevant streptogramins quinupristin and dalfopristin reveals their unique inhibitory mechanism. Quinupristin, a streptogramin B compound, binds in the ribosomal exit tunnel in a similar manner and position as the macrolides, suggesting a similar inhibitory mechanism, namely blockage of the ribosomal tunnel. Dalfopristin, the corresponding streptogramin A compound, binds close to quinupristin directly within the peptidyl transferase centre affecting both A- and P-site occupation by tRNA molecules.ConclusionsThe crystal structure indicates that the synergistic effect derives from direct interaction between both compounds and shared contacts with a single nucleotide, A2062. Upon binding of the streptogramins, the peptidyl transferase centre undergoes a significant conformational transition, which leads to a stable, non-productive orientation of the universally conserved U2585. Mutations of this rRNA base are known to yield dominant lethal phenotypes. It seems, therefore, plausible to conclude that the conformational change within the peptidyl transferase centre is mainly responsible for the bactericidal activity of the streptogramins and the post-antibiotic inhibition of protein synthesis.

X-ray crystallography study on ribosome recycling: the mechanism of binding and action of RRF on the 50S ribosomal subunit

This study presents the crystal structure of domain I of the Escherichia coli ribosome recycling factor (RRF) bound to the Deinococcus radiodurans 50S subunit. The orientation of RRF is consistent with the position determined on a 70S‐RRF complex by cryoelectron microscopy (cryo‐EM). Alignment, however, requires a rotation of 7° and a shift of the cryo‐EM RRF by a complete turn of an α‐helix, redefining the contacts established with ribosomal components. At 3.3 Å resolution, RRF is seen to interact exclusively with ribosomal elements associated with tRNA binding and/or translocation. Furthermore, these results now provide a high‐resolution structural description of the conformational changes that were suspected to occur on the 70S‐RRF complex, which has implications for the synergistic action of RRF with elongation factor G (EF‐G). Specifically, the tip of the universal bridge element H69 is shifted by 20 Å toward h44 of the 30S subunit, suggesting that RRF primes the intersubunit bridge B2a for the action of EF‐G. Collectively, our data enable a model to be proposed for the dual action of EF‐G and RRF during ribosome recycling.

High heterogeneity within the ribosomal proteins of the Arabidopsis thaliana 80S ribosome

Proteomic studies have addressed the composition of plant chloroplast ribosomes and 70S ribosomes from the unicellular organism Chlamydomonas reinhardtii But comprehensive characterization of cytoplasmic 80S ribosomes from higher plants has been lacking. We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to analyse the cytoplasmic 80S ribosomes from the model flowering plant Arabidopsis thaliana. Of the 80 ribosomal protein families predicted to comprise the cytoplasmic 80S ribosome, we have confirmed the presence of 61; specifically, 27 (84%) of the small 40S subunit and 34 (71%) of the large 60S subunit. Nearly half (45%) of the ribosomal proteins identified are represented by two or more distinct spots in the 2-DE gel indicating that these proteins are either post-translationally modified or present as different isoforms. Consistently, MS-based protein identification revealed that at least one-third (34%) of the identified ribosomal protein families showed expression of two or more family members. In addition, we have identified a number of non-ribosomal proteins that co-migrate with the plant 80S ribosomes during gradient centrifugation suggesting their possible association with the 80S ribosomes. Among them, RACK1 has recently been proposed to be a ribosome-associated protein that promotes efficient translation in yeast. The study, thus provides the basis for further investigation into the function of the other identified non-ribosomal proteins as well as the biological meaning of the various ribosomal protein isoforms.

Three-dimensional structures of translating ribosomes by Cryo-EM

Cryo-electron microscopy and image reconstruction techniques have been used to obtain three-dimensional maps for E. coli ribosomes stalled following translation of three representative proteins. Comparisons of these electron density maps, at resolutions of between 13 and 16 A, with that of a nontranslating ribosome pinpoint specific structural differences in stalled ribosomes and identify additional material, including tRNAs and mRNA. In addition, the tunnel through the large subunit, the anticipated exit route of newly synthesized proteins, is partially occluded in all the stalled ribosome structures. This observation suggests that significant segments of the nascent polypeptide chains examined here could be located within an expanded tunnel, perhaps in a rudimentary globular conformation. Such behavior could be an important aspect of the folding of at least some proteins in the cellular environment.

Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy

Protein folding in living cells is inherently coupled to protein synthesis and chain elongation. There is considerable evidence that some nascent chains fold into their native structures in a cotranslational manner before release from the ribosome, but, despite its importance, a detailed description of such a process at the atomic level remains elusive. We show here at a residue-specific level that a nascent protein chain can reach its native tertiary structure on the ribosome. By generating translation-arrested ribosomes in which the newly synthesized polypeptide chain is selectively 13C/15N-labeled, we observe, using ultrafast NMR techniques, a large number of resonances of a ribosome-bound nascent chain complex corresponding to a pair of C-terminally truncated immunoglobulin (Ig) domains. Analysis of these spectra reveals that the nascent chain adopts a structure in which a native-like N-terminal Ig domain is tethered to the ribosome by a largely unfolded and highly flexible C-terminal domain. Selective broadening of resonances for a group of residues that are colocalized in the structure demonstrates that there are specific but transient interactions between the ribosome and the N-terminal region of the folded Ig domain. These findings represent a step toward a detailed structural understanding of the cellular processes of cotranslational folding.