Gulnara Yusupova

New structural insights into the decoding mechanism: Translation infidelity via a G·U pair with Watson–Crick geometry

Pioneer crystallographic studies of the isolated 30S ribosomal subunit provided the first structural insights into the decoding process. Recently, new crystallographic data on full 70S ribosomes with mRNA and tRNAs have shown that the formation of the tight decoding centre is ensured by conformational rearrangement of the 30S subunit (domain closure), which is identical for cognate or near‐cognate tRNA. When a G·U forms at the first or second codon–anticodon positions (near‐cognate tRNA), the ribosomal decoding centre forces the adoption of Watson–Crick G·C‐like geometry rather than that of the expected Watson–Crick wobble pair. Energy expenditure for rarely occuring tautomeric base required for Watson–Crick G·C‐like G·U pair or the repulsion energy due to steric clash within the mismatched base pair could constitute the only cause for efficient rejection of a near‐cognate tRNA. Our data suggest that “geometrical mimicry” can explain how wrong aminoacyl‐tRNAs with G·U pairs in the codon–anticodon helix forming base pairs with Watson–Crick geometry in the decoding center can be incorporated into the polypeptide chain.

Ribosomal Initiation Complexes Probed by Toeprinting and Effect of trans-Acting Translational Regulators in Bacteria

Toeprinting was developed to study the formation of ribosomal initiation complexes in bacteria. This approach, based on the inhibition of reverse transcriptase elongation, was used to monitor the effect of ribosomal components and translational factors on the formation of the active ribosomal initiation complex. Moreover, this method offers an easy way to study in vitro how mRNA conformational changes alter ribosome binding at the initiation site. These changes can be induced either by environmental cues (temperature, ion concentration), or by the binding of metabolites, regulatory proteins, and trans-acting RNAs. An experimental guide is given to follow the different steps of the formation of ribosomal initiation complexes in Escherichia coli and Staphylococcus aureus, and to monitor the mechanism of action of several regulators on translation initiation in vitro. Protocols to prepare the ribosome and the subunits are also given for Thermus thermophilus, Staphylococcus aureus, and Escherichia coli.

New Structural Insights into Translational Miscoding

The fidelity of translation depends strongly on the selection of the correct aminoacyl-tRNA that is complementary to the mRNA codon present in the ribosomal decoding center. The ribosome occasionally makes mistakes by selecting the wrong substrate from the pool of aminoacyl-tRNAs. Here, we summarize recent structural advances that may help to clarify the origin of missense errors that occur during decoding. These developments suggest that discrimination between tRNAs is based primarily on steric complementarity and shape acceptance rather than on the number of hydrogen bonds between the molding of the decoding center and the codon-anticodon duplex. They strengthen the hypothesis that spatial mimicry, due either to base tautomerism or ionization, drives infidelity in ribosomal translation.

Structures and dynamics of hibernating ribosomes from Staphylococcus aureus mediated by intermolecular interactions of HPF

In bacteria, ribosomal hibernation shuts down translation as a response to stress, through reversible binding of stress‐induced proteins to ribosomes. This process typically involves the formation of 100S ribosome dimers. Here, we present the structures of hibernating ribosomes from human pathogen Staphylococcus aureus containing a long variant of the hibernation‐promoting factor (SaHPF) that we solved using cryo‐electron microscopy. Our reconstructions reveal that the N‐terminal domain (NTD) of SaHPF binds to the 30S subunit as observed for shorter variants of HPF in other species. The C‐terminal domain (CTD) of SaHPF protrudes out of each ribosome in order to mediate dimerization. Using NMR, we characterized the interactions at the CTD‐dimer interface. Secondary interactions are provided by helix 26 of the 16S ribosomal RNA. We also show that ribosomes in the 100S particle adopt both rotated and unrotated conformations. Overall, our work illustrates a specific mode of ribosome dimerization by long HPF, a finding that may help improve the selectivity of antimicrobials.

The ribosome prohibits the G•U wobble geometry at the first position of the codon-anticodon helix.

Precise conversion of genetic information into proteins is essential to cellular health. However, a margin of error exists and is at its highest on the stage of translation of mRNA by the ribosome. Here we present three crystal structures of 70S ribosome complexes with messenger RNA and transfer RNAs and show that when a G•U base pair is at the first position of the codon–anticodon helix a conventional wobble pair cannot form because of inescapable steric clash between the guanosine of the A codon and the key nucleotide of decoding center adenosine 1493 of 16S rRNA. In our structure the rigid ribosomal decoding center, which is identically shaped for cognate or near-cognate tRNAs, forces this pair to adopt a geometry close to that of a canonical G•C pair. We further strengthen our hypothesis that spatial mimicry due either to base tautomerism or ionization dominates the translation infidelity mechanism.

Structure of the 70S ribosome from human pathogen Staphylococcus aureus.

Comparative structural studies of ribosomes from various organisms keep offering exciting insights on how species-specific or environment-related structural features of ribosomes may impact translation specificity and its regulation. Although the importance of such features may be less obvious within more closely related organisms, their existence could account for vital yet species-specific mechanisms of translation regulation that would involve stalling, cell survival and antibiotic resistance. Here, we present the first full 70S ribosome structure from Staphylococcus aureus, a Gram-positive pathogenic bacterium, solved by cryo-electron microscopy. Comparative analysis with other known bacterial ribosomes pinpoints several unique features specific to S. aureus around a conserved core, at both the protein and the RNA levels. Our work provides the structural basis for the many studies aiming at understanding translation regulation in S. aureus and for designing drugs against this often multi-resistant pathogen.

Crystal structure of eukaryotic ribosome and its complexes with inhibitors.

A high-resolution structure of the eukaryotic ribosome has been determined and has led to increased interest in studying protein biosynthesis and regulation of biosynthesis in cells. The functional complexes of the ribosome crystals obtained from bacteria and yeast have permitted researchers to identify the precise residue positions in different states of ribosome function. This knowledge, together with electron microscopy studies, enhances our understanding of how basic ribosome processes, including mRNA decoding, peptide bond formation, mRNA, and tRNA translocation and cotranslational transport of the nascent peptide, are regulated. In this review, we discuss the crystal structure of the entire 80S ribosome from yeast, which reveals its eukaryotic-specific features, and application of X-ray crystallography of the 80S ribosome for investigation of the binding mode for distinct compounds known to inhibit or modulate the protein-translation function of the ribosome. We also refer to a challenging aspect of the structural study of ribosomes, from higher eukaryotes, where the structures of major distinctive features of higher eukaryote ribosome—the high-eukaryote–specific long ribosomal RNA segments (about 1MDa)—remain unresolved. Presently, the structures of the major part of these high-eukaryotic expansion ribosomal RNA segments still remain unresolved. This article is part of the themed issue ‘Perspectives on the ribosome’.

Aminoglycoside interactions and impacts on the eukaryotic ribosome.

Significance Aminoglycosides are well known as antibiotics that target the bacterial ribosome. However, they also impact the eukaryotic translation mechanism to promote read-through of premature termination codons (PTCs) in mRNA. Aminoglycosides are therefore considered as potential therapies for PTC-associated human diseases. Here, we performed a comprehensive study of the mechanism of action of aminoglycosides in eukaryotes by applying a combination of structural and functional approaches. Our findings reveal complex interactions of aminoglycosides with eukaryotic 80S ribosome caused by their multiple binding sites, which lead to inhibition of intersubunit movement within the human ribosome that impact nearly every aspect of protein synthesis. 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-Å 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.

The Amaryllidaceae Alkaloid Haemanthamine Binds the Eukaryotic Ribosome to Repress Cancer Cell Growth.

Alkaloids isolated from the Amaryllidaceae plants have potential as therapeutics for treating human diseases. Haemanthamine has been studied as a novel anticancer agent due to its ability to overcome cancer cell resistance to apoptosis. Biochemical experiments have suggested that hemanthamine targets the ribosome. However, a structural characterization of its mechanism has been missing. Here we present the 3.1xa0Å resolution X-ray structure of haemanthamine bound to the Saccharomyces cerevisiae 80S ribosome. This structure reveals that haemanthamine targets the A-site cleft on the large ribosomal subunit rearranging rRNA to halt the elongation phase of translation. Furthermore, we provide evidence that haemanthamine and other Amaryllidaceae alkaloids also inhibit specifically ribosome biogenesis, triggering nucleolar stress response and leading to p53 stabilization in cancer cells. Together with a computer-aided interpretation of existing structure-activity relationships of Amaryllidaceae alkaloids congeners, we provide a rationale for designing molecules with enhanced potencies and reduced toxicities.

X-Ray Analysis of Prokaryotic and Eukaryotic Ribosomes

The ribosome is the giant ribonucleoprotein assembly that translates the genetic code into protein in all living cells. Ribosomes from bacteria and archaea consist of a large (50S) and a small (30S) subunit, which together compose the 2.5 megadalton (MDa) 70S ribosome. Their eukaryotic counterparts are the 60S and 40S subunits and the 80S ribosome (from 3.5 MDa in lower eukaryotes to 4.5 MDa in higher). Many ribosomal key components are conserved across the three kingdoms of life: bacteria, archaea, and eukarya and constitutes a common core undertaking the fundamental processes of protein biosynthesis. The process of protein synthesis has been studied during the last five decades, and for most of this time, information of the three-dimensional structure of the ribosome has been vague and sparse. Cryo-electron microscopy and single-particle analysis produced the first direct visualizations of the bacterial ribosome in different functional states (Frank et al. 1995; Stark et al. 1997a, b; Agrawal et al. 1998). However, not until the X-ray crystallographic structures of the entire 70S ribosome as well as the individual 30S and 50S subunits began to emerge did accurate atomic models become available.