Marat Yusupov

The Structure of the Eukaryotic Ribosome at 3.0 Å Resolution

A close-up view of the ribosome’s 79 proteins and 5500 RNA nucleotides. Ribosomes translate genetic information encoded by messenger RNA into proteins. Many aspects of translation and its regulation are specific to eukaryotes, whose ribosomes are much larger and intricate than their bacterial counterparts. We report the crystal structure of the 80S ribosome from the yeast Saccharomyces cerevisiae—including nearly all ribosomal RNA bases and protein side chains as well as an additional protein, Stm1—at a resolution of 3.0 angstroms. This atomic model reveals the architecture of eukaryote-specific elements and their interaction with the universally conserved core, and describes all eukaryote-specific bridges between the two ribosomal subunits. It forms the structural framework for the design and analysis of experiments that explore the eukaryotic translation apparatus and the evolutionary forces that shaped it.

The path of messenger RNA through the ribosome.

Using X-ray crystallography, we have directly observed the path of mRNA in the 70S ribosome in Fourier difference maps at 7 A resolution. About 30 nucleotides of the mRNA are wrapped in a groove that encircles the neck of the 30S subunit. The Shine-Dalgarno helix is bound in a large cleft between the head and the back of the platform. At the interface, only about eight nucleotides (-1 to +7), centered on the junction between the A and P codons, are exposed, and bond almost exclusively to 16S rRNA. The mRNA enters the ribosome around position +13 to +15, the location of downstream pseudoknots that stimulate -1 translational frame shifting.

Crystal Structure of the Eukaryotic Ribosome

Macromolecular Message Translation The ribosome is a macromolecular machine that translates the sequence of messenger RNA into proteins in all living cells. Structures of prokaryotic ribosomes have supplied insight into the conserved features of such protein synthesis; however, eukaryotic translation has additional levels of complexity. Ben-Shem et al. (p. 1203) have determined the crystal structure of the yeast 80S ribosome at 4.15 angstrom resolution. The ribosome is in a ratcheted conformation, which is a state that is an intermediate in the translocation of messenger RNA and transfer RNA. The crystal structure provides the molecular underpinning for existing biochemical and genetic data and will inform the design of functional experiments. The structure of the 80S ribosome from yeast has been determined at 4.15 angstrom resolution. Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation.

Structural basis for messenger RNA movement on the ribosome

Translation initiation is a major determinant of the overall expression level of a gene. The translation of functionally active protein requires the messenger RNA to be positioned on the ribosome such that the start/initiation codon will be read first and in the correct frame. Little is known about the molecular basis for the interaction of mRNA with the ribosome at different states of translation. Recent crystal structures of the ribosomal subunits, the empty 70S ribosome and the 70S ribosome containing functional ligands have provided information about the general organization of the ribosome and its functional centres. Here we compare the X-ray structures of eight ribosome complexes modelling the translation initiation, post-initiation and elongation states. In the initiation and post-initiation complexes, the presence of the Shine–Dalgarno (SD) duplex causes strong anchoring of the 5′-end of mRNA onto the platform of the 30S subunit, with numerous interactions between mRNA and the ribosome. Conversely, the 5′ end of the ‘elongator’ mRNA lacking SD interactions is flexible, suggesting a different exit path for mRNA during elongation. After the initiation of translation, but while an SD interaction is still present, mRNA moves in the 3′→5′ direction with simultaneous clockwise rotation and lengthening of the SD duplex, bringing it into contact with ribosomal protein S2.

A new understanding of the decoding principle on the ribosome.

During protein synthesis, the ribosome accurately selects transfer RNAs (tRNAs) in accordance with the messenger RNA (mRNA) triplet in the decoding centre. tRNA selection is initiated by elongation factor Tu, which delivers tRNA to the aminoacyl tRNA-binding site (A site) and hydrolyses GTP upon establishing codon–anticodon interactions in the decoding centre. At the following proofreading step the ribosome re-examines the tRNA and rejects it if it does not match the A codon. It was suggested that universally conserved G530, A1492 and A1493 of 16S ribosomal RNA, critical for tRNA binding in the A site, actively monitor cognate tRNA, and that recognition of the correct codon–anticodon duplex induces an overall ribosome conformational change (domain closure). Here we propose an integrated mechanism for decoding based on six X-ray structures of the 70S ribosome determined at 3.1–3.4 Å resolution, modelling cognate or near-cognate states of the decoding centre at the proofreading step. We show that the 30S subunit undergoes an identical domain closure upon binding of either cognate or near-cognate tRNA. This conformational change of the 30S subunit forms a decoding centre that constrains the mRNA in such a way that the first two nucleotides of the A codon are limited to form Watson–Crick base pairs. When U·G and G·U mismatches, generally considered to form wobble base pairs, are at the first or second codon–anticodon position, the decoding centre forces this pair to adopt the geometry close to that of a canonical C·G pair. This by itself, or with distortions in the codon–anticodon mini-helix and the anticodon loop, causes the near-cognate tRNA to dissociate from the ribosome.

One core, two shells: bacterial and eukaryotic ribosomes

Ribosomes are universally conserved enzymes that carry out protein biosynthesis. Bacterial and eukaryotic ribosomes, which share an evolutionarily conserved core, are thought to have evolved from a common ancestor by addition of proteins and RNA that bestow different functionalities to ribosomes from different domains of life. Recently, structures of the eukaryotic ribosome, determined by X-ray crystallography, have allowed us to compare these structures to previously determined structures of bacterial ribosomes. Here we describe selected bacteria- or eukaryote-specific structural features of the ribosome and discuss the functional implications of some of them.

Structural basis for the inhibition of the eukaryotic ribosome

The ribosome is a molecular machine responsible for protein synthesis and a major target for small-molecule inhibitors. Compared to the wealth of structural information available on ribosome-targeting antibiotics in bacteria, our understanding of the binding mode of ribosome inhibitors in eukaryotes is currently limited. Here we used X-ray crystallography to determine 16 high-resolution structures of 80S ribosomes from Saccharomyces cerevisiae in complexes with 12 eukaryote-specific and 4 broad-spectrum inhibitors. All inhibitors were found associated with messenger RNA and transfer RNA binding sites. In combination with kinetic experiments, the structures suggest a model for the action of cycloheximide and lactimidomycin, which explains why lactimidomycin, the larger compound, specifically targets the first elongation cycle. The study defines common principles of targeting and resistance, provides insights into translation inhibitor mode of action and reveals the structural determinants responsible for species selectivity which could guide future drug development.

Translocation of tRNA during protein synthesis

Coupled translocation of tRNA and mRNA in the ribosome during protein synthesis is one of the most challenging and intriguing problems in the field of translation. We highlight several key questions regarding the mechanism of translocation, and discuss possible mechanistic models in light of the recent crystal structures of the ribosome and its subunits.

Crystallization of 70 S ribosomes and 30 S ribosomal subunits from Thermus thermophilus

Well‐ordered three‐dimensional crystals of 70 S ribosomes and 30 S ribosomal subunits from extremely thermophilic bacteria Thermus thermophilus have been obtained. Positively stained thin sections of the crystals have been analyzed by electron microscopy. Redissolved crystalline ribosomes and small ribosomal subunits reveal sedimentation constants of 70 S and 30 S, respectively, and are functionally active in the poly(U)‐system.

Crystal structure of the 80S yeast ribosome.

The first X-ray structure of the eukaryotic ribosome at 3.0Å resolution was determined using ribosomes isolated and crystallized from the yeast Saccharomyces cerevisiae (Ben-Shem A, Garreau de Loubresse N, Melnikov S, Jenner L, Yusupova G, Yusupov M: The structure of the eukaryotic ribosome at 3.0 A resolution. Science 2011, 334:1524-1529). This accomplishment was possible due to progress in yeast ribosome biochemistry as well as recent advances in crystallographic methods developed for structure determination of prokaryotic ribosomes isolated from Thermus thermophilus and Escherichia coli. In this review we will focus on the development of isolation procedures that allowed structure determination (both cryo-EM and X-ray crystallography) to be successful for the yeast S. cerevisiae. Additionally we will introduce a new nomenclature that facilitates comparison of ribosomes from different species and kingdoms of life. Finally we will discuss the impact of the yeast 80S ribosome crystal structure on perspectives for future investigations.