Search

Thomas A. Steitz was among the foremost of the generation that was responsible for an explosion in our understanding of the structure and function of biological macromolecules. His research career was one of sustained excellence over six decades, and spanned the range from determining the structures of important metabolic enzymes to understanding the structural basis of how genetic information residing in our DNA is used to make the proteins they encode. This latter effort culminated in the structure of the ribosome, for which he shared the Nobel Prize in Chemistry in 2009.

Finding the start Eukaryotic translation involves many players in a dynamic and well-orchestrated process. A 43 S preinitiation complex (PIC) comprises the 40 S ribosomal subunit; initiation factors, including the eIF3 complex, which is known to play a key role; and the transfer RNA used for translation initiation. The PIC is recruited to the cap-binding complex eIF4F at the 5′ end of messenger RNA (mRNA) to form a 48 S complex that scans along the mRNA for a start codon. Brito Querido et al. determined the structure of a reconstituted human 48 S complex using cryo–electron microscopy. They found that eIF4F binds to eIF3 near the exit site of the ribosome. This positioning suggests that downstream mRNA is likely pulled through the 40 S subunit to find the start codon. Science , this issue p. 1220

Funder: Agouron Institute, Funder: Louis-Jeantet Foundation, Ribosome assembly is an essential and conserved process that is regulated at each step by specific factors. Using cryo-electron microscopy (cryo-EM), we visualize the formation of the conserved peptidyl transferase center (PTC) of the human mitochondrial ribosome. The conserved GTPase GTPBP7 regulates the correct folding of 16S ribosomal RNA (rRNA) helices and ensures 2'-O-methylation of the PTC base U3039. GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which sequester H68-71 of the 16S rRNA and allow biogenesis factors to access the maturing PTC. Mutations that disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activate mitochondrial stress and cause viability, development, and sterility defects. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4, but not its enzymatic activity, is indispensable for mitochondrial protein synthesis.

Quality control in mitochondria Human mitochondria have their own genome and ribosomes called mitoribosomes that respectively encode and synthesize essential subunits of complexes that use the energy from the oxidation of metabolites to drive the synthesis of adenosine triphosphate (ATP). These complexes are key to the health of the cell. Desai et al. studied a mitoribosome-associated quality control pathway that prevents aberrant translation. They purified mitoribosomes under conditions designed to induce stalling and determined the structures of two intermediates in the rescue pathway. These structures revealed two proteins that eject the unfinished polypeptide chain and peptidyl transfer RNA from the ribosome. Their cryo–electron microscopy dataset also revealed additional states that may correspond to intermediates in the mitochondrial translation elongation cycle. Science , this issue p. 1105

I want to start by congratulating my former colleague Greg Winter on his Nobel Prize, as well as the winners of this year's Medals and Awards. It is also with great sadness that I note that Aaron Klug, who was president from 1995 to 2000, died 10 days ago.

In this interview Venki Ramakrishnan reviews part of his work on the structural resolution of the ribosome, for which he was jointly awarded the Nobel Prize for Chemistry in 2009. He discusses the role that synchrotron facilities have played in unravelling the structure of the ribosome and how cryogenic electron microscopy (cryo-EM) has become an essential tool for structural biologists. He concludes with an overview on his current research activities at the MRC Laboratory of Molecular Biology.

Venkatraman ‘Venki’ Ramakrishnan is the President of The Royal Society and Group Leader at the MRC Laboratory of Molecular Biology. In 2009 he shared the Nobel Prize in Chemistry ‘for studies of the structure and function of the ribosome’. In this interview he explains why governments should invest more in basic scientific research rather than simply on applied science and engineering. He also discusses interdisciplinarity, collaborations, and public engagement.

Mammalian mitochondrial ribosomes (mitoribosomes) have less rRNA content and 36 additional proteins compared with the evolutionarily related bacterial ribosome. These differences make the assembly of mitoribosomes more complex than the assembly of bacterial ribosomes, but the molecular details of mitoribosomal biogenesis remain elusive. Here, we report the structures of two late-stage assembly intermediates of the human mitoribosomal large subunit (mt-LSU) isolated from a native pool within a human cell line and solved by cryo-EM to approximately 3-A resolution. Comparison of the structures reveals insights into the timing of rRNA folding and protein incorporation during the final steps of ribosomal maturation and the evolutionary adaptations that are required to preserve biogenesis after the structural diversification of mitoribosomes. Furthermore, the structures redefine the ribosome silencing factor (RsfS) family as multifunctional biogenesis factors and identify two new assembly factors (L0R8F8 and mt-ACP) not previously implicated in mitoribosomal biogenesis.

An international commission from the US National Academy of Sciences, the US National Academy of Medicine and the UK Royal Society.

Rescuing stalled ribosomes A small percentage of bacterial mRNAs lack a stop codon. Ribosomes stall at the end of such mRNAs, and the buildup of stalled ribosomes can be lethal. The primary rescue mechanism, in which translation continues on a piece of RNA that contains a stop codon, is a drug target. However, bacteria have another backup plan. James et al. present structures that show that ArfA (alternative rescue factor A) substitutes for a stop codon by binding in the ribosomal mRNA channel and recruiting RF2 (release factor 2). It mediates conformational changes required for RF2 to catalyze peptide release. Science , this issue p. 1437

In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation that leads to rapid and comprehensive reprogramming of metabolic and transcriptional patterns. In general, transcription of genes for growth and proliferation is downregulated, while those important for survival and virulence are upregulated. Amino acid starvation is sensed by depletion of the aminoacylated tRNA pools, and this results in accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA in the ribosomal A site. RelA is recruited to stalled ribosomes and activated to synthesize a hyperphosphorylated guanosine analogue, (p)ppGpp, which acts as a pleiotropic secondary messenger. However, structural information about how RelA recognizes stalled ribosomes and discriminates against aminoacylated tRNAs is missing. Here we present the cryo-electron microscopy structure of RelA bound to the bacterial ribosome stalled with uncharged tRNA. The structure reveals that RelA utilizes a distinct binding site compared to the translational factors, with a multi-domain architecture that wraps around a highly distorted A-site tRNA. The TGS (ThrRS, GTPase and SpoT) domain of RelA binds the CCA tail to orient the free 3' hydroxyl group of the terminal adenosine towards a β-strand, such that an aminoacylated tRNA at this position would be sterically precluded. The structure supports a model in which association of RelA with the ribosome suppresses auto-inhibition to activate synthesis of (p)ppGpp and initiate the stringent response. Since stringent control is responsible for the survival of pathogenic bacteria under stress conditions, and contributes to chronic infections and antibiotic tolerance, RelA represents a good target for the development of novel antibacterial therapeutics.

11 páginas, 5 figuras, 1 tabla., One of the responses to stress by eukaryotic cells is the down-regulation of protein synthesis by phosphorylation of translation initiation factor eIF2. Phosphorylation results in low availability of the eIF2 ternary complex (eIF2-GTP-tRNAi) by affecting the interaction of eIF2 with its GTP-GDP exchange factor eIF2B. We have determined the cryo-EM structure of yeast eIF2B in complex with phosphorylated eIF2 at an overall resolution of 4.2 Å. Two eIF2 molecules bind opposite sides of an eIF2B hetero-decamer through eIF2α-D1, which contains the phosphorylated Ser51. eIF2α-D1 is mainly inserted between the N-terminal helix bundle domains of δ and α subunits of eIF2B. Phosphorylation of Ser51 enhances binding to eIF2B through direct interactions of phosphate groups with residues in eIF2Bα and indirectly by inducing contacts of eIF2α helix 58-63 with eIF2Bδ leading to a competition with Met-tRNAi., This study was supported by the MRC-LMB EM Facility. This work was supported by grants from the Medical Research Council (MC_U105184332) and the Wellcome Trust (WT096570) to V.R. and by a grant BFU2017-85814-P from the Spanish government to J.L.L.

Mechanism of ribosome rescue Bacterial ribosomes that stall on truncated or cleaved messenger RNA (mRNA) are rescued by trans-translation. Two factors, transfer-messenger RNA (tmRNA) and small protein B (SmpB), resolve the stalled complex by tagging the nascent polypeptide for degradation and facilitating release of the ribosome. Rae et al. determined structures of key trans-translation intermediates. The structures reveal how SmpB identifies stalled ribosomes; how the large, circularized tmRNA molecule moves through the ribosome; and how translation is shifted from the truncated mRNA to tmRNA. Science , this issue p. 740

During translational initiation in eukaryotes, the 40S ribosomal subunit forms a 48S pre-initiation complex (PIC) with eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAMeti) and eIF5. The 48S PIC, in an open conformation, scans the 5' untranslated region of mRNA until it encounters a start codon in the P site. We present a single particle cryo-electron microscopy (cryo-EM) reconstruction of a 48S PICs from yeast in an open scanning-competent state at 5.15[A] resolution. The structure contains eIF3 PCI domains bound on the solvent side of the 40S, that was not observed in the earlier reported complex in an open state. eIF3b is observed bound on the 40S subunit interface, re-located with eIF3i from their solvent-interface locations observed in other PIC structures; however, the eIF3i {beta}-propeller is not in contact with the 40S. We also present a map obtained by masked classification with signal subtraction around the observed {beta}-propeller of eIF3b on the intersubunit interface of our previous 48S PICs in closed state, which also shows similar re-location of eIF3b and eIF3i from the solvent interface. Overall, the structures reported here are similar to those reported earlier but the relatively higher resolution of 48S PIC-open and the higher local resolution around the eIF3 {beta}-propellers in py48S-closed enabled a more detailed assignment of eIF3 subunits and revealed relocation of the entire quaternary complex of eIF3b/eIF3i/eIF3g/eIF3a-Cter to the intersubunit interface of 40S.

33 páginas, 10 figuras, 5 tablas, In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNAi in a 'PIN' state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a yeast 48S pre-initiation complex (PIC), at an overall resolution of 3.0 Å, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG codonsin vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection., We are grateful to CG Savva, G McMullan for technical support with cryo-EM, T Darling and J Grimmett for help with computing and J Brasa for help with figures/movies. JL was supported by a FEBS postdoctoral fellowship. TH acknowledges start-up funds from the Indian Institute of Science. This work was funded by grants to from the UK Medical Research Council (MC_U105184332), Wellcome Trust Senior Investigator award (WT096570), the Agouron Institute and the Jeantet Foundation to VR; by the Department of Science and Technology, Government of India Grant [Int/NZ/P-2/13] to AKS; from the NIH (GM62128) formerly to JRL; the Human Frontiers in Science Program (RGP-0028/ 2009) to AGH, JRL and VR; and by the Intramural Research Program of the NIH (AGH, JRL).

Summary Aberrantly slow translation elicits quality control pathways initiated by the ubiquitin ligase ZNF598. How ZNF598 discriminates physiologic from pathologic translation complexes and ubiquitinates stalled ribosomes selectively is unclear. Here, we find that the minimal unit engaged by ZNF598 is the collided di-ribosome, a molecular species that arises when a trailing ribosome encounters a slower leading ribosome. The collided di-ribosome structure reveals an extensive 40S-40S interface in which the ubiquitination targets of ZNF598 reside. The paucity of 60S interactions allows for different ribosome rotation states, explaining why ZNF598 recognition is indifferent to how the leading ribosome has stalled. The use of ribosome collisions as a proxy for stalling allows the degree of tolerable slowdown to be tuned by the initiation rate on that mRNA; hence, the threshold for triggering quality control is substrate specific. These findings illustrate how higher-order ribosome architecture can be exploited by cellular factors to monitor translation status., Graphical Abstract, Highlights • ZNF598 is a direct sensor of ribosome collisions incurred by many unrelated causes • The minimal target recognized and ubiquitinated by ZNF598 is a collided di-ribosome • Collided di-ribosome structure shows that ZNF598 ubiquitin sites are near the interface • Collisions are required to terminally arrest translation in ZNF598-dependent manner, Cells contain millions of ribosomes that need to translate mRNAs accurately to maintain homeostasis. Ribosomes that slow excessively during translation must be promptly resolved to avoid disease. Juszkiewicz et al. show that ribosome collisions, a sign of aberrant translation, are detected by the ubiquitin ligase ZNF598 to initiate quality control.

Summary Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5′ end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition., Graphical Abstract, Highlights • Structures of eukaryotic translation initiation complexes in open and closed states • In the open complex the 40S head moves upward to open the mRNA entry channel latch • Transition to closed state locks initiator tRNA in the P site base-paired with AUG • The structures show how eIF3 contacts eIF2 and eIF1 on the 40S subunit interface, The small ribosomal subunit latch has to open to allow mRNA loading. Llacer et al. report cryo-EM reconstructions of yeast translation pre-initiation complexes in both open and closed conformations, highlighting structural differences and elucidating key events in start codon recognition.

Clarity in UK and EU policy must come soon, so science funds can be spent well, says Venki Ramakrishnan. Clarity in UK and EU policy must come soon, so science funds can be spent well, says Venki Ramakrishnan.

Making mitochondrial hydrophobic proteins Mitochondria produce chemical energy for the cell. Human mitochondria have their own specific ribosomes—mitoribosomes, which are distinct from cytoplasmic ribosomes. Mitoribosomes synthesize the mitochondrial membrane proteins that generate the chemical energy. Brown et al. used cryo–electron microscopy to determine the high-resolution structure of the large subunit of the human mitoribosome. The mitoribosome has a number of unique features, including an exit tunnel lined with hydrophobic amino acid residues. Science , this issue p. 718

6 páginas, 6 figuras. Contiete material suplementario, Mitochondria have specialized ribosomes that have diverged from their bacterial and cytoplasmic counterparts. We have solved the structure of the yeast mitoribosomal large subunit using single-particle cryo-electron microscopy. The resolution of 3.2 angstroms enabled a nearly complete atomic model to be built de novo and refined, including 39 proteins, 13 of which are unique to mitochondria, as well as expansion segments of mitoribosomal RNA. The structure reveals a new exit tunnel path and architecture, unique elements of the E site, and a putative membrane docking site., This work was funded by grants from the UK Medical Research Council (MC_U105184332 to VR, MC_UP_A025_1013 to SHWS and MC_UP_A025_1012 to GM); a Wellcome Trust Senior Investigator award (WT096570), the Agouron Institute and the Jeantet Foundation (VR); fellowships from Human Frontiers Science Programme (AA), EU FP7 Marie Curie (XB), FEBS (JLL), and EMBO (TH). Cryo-EM density maps have been deposited with the EMDB (accession number EMD-2566) and coordinates have been deposited with the PDB (entry code 3J6B).

A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as ‘new system’ names.

The bacterial stringent response links nutrient starvation with the transcriptional control of genes. This process is initiated by the stringent factor RelA, which senses the presence of deacylated tRNA in the ribosome as a symptom of amino-acid starvation to synthesize the alarmone (p)ppGpp. Here we report a cryo-EM study of RelA bound to ribosomes bearing cognate, deacylated tRNA in the A-site. The data show that RelA on the ribosome stabilizes an unusual distorted form of the tRNA, with the acceptor arm making contact with RelA and far from its normal location in the peptidyl transferase centre.

The yeast mitoribosome Mitochondria are eukaryotic organelles that produce ATP, the energy source of the cell. They have dedicated ribosomes (mitoribosomes) that encode some of the membrane proteins that are essential to ATP production. Desai et al. present a high-resolution structure of the 75-component yeast mitoribosome, determined by electron cryomicroscopy. Mitoribosomes share an ancestor with modern bacterial ribosomes. Comparing the structure of the yeast mitoribosome with mammalian mitoribosomes suggests how they have evolved differently to perform species-specific functions. Science , this issue p. 528

Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2). The CrPV-IRES restricts the otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). Once the 60S is recruited, the binary CrPV-IRES/80S complex oscillates between canonical and rotated states (Fernández et al., 2014; Koh et al., 2014), as seen for pre-translocation complexes with tRNAs. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. Key residues in domain IV of eEF2 interact with pseudoknot I (PKI) of the CrPV-IRES stabilizing it in a conformation reminiscent of a hybrid tRNA state. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation.

Protein release factor 3 (RF3), a guanosine triphosphatase, binds to ribosome after release of the nascent peptide and promotes dissociation of the class I release factors during the termination of protein synthesis. Here we present the crystal structure of the 70S ribosome with RF3 in the presence of a nonhydrolyzable GTP analogue, guanosine 5′-β,γ-methylenetriphosphate (GDPCP), refined to 3.8 Å resolution. The structure shows that the subunits of the ribosome are rotated relative to each other compared to the canonical state, resulting in a P/E hybrid state for the transfer RNA. The substantial conformational rearrangements in the complex are described and suggest how RF3, by stabilizing the hybrid state of the ribosome, facilitates the dissociation of class I release factors.

The ribosome converts genetic information into protein by selecting aminoacyl tRNAs whose anticodons base-pair to an mRNA codon. Mutations in the tRNA body can perturb this process and affect fidelity. The Hirsh suppressor is a well-studied tRNA^(Trp) harboring a G24A mutation that allows readthrough of UGA stop codons. Here we present crystal structures of the 70S ribosome complexed with EF-Tu and aminoacyl tRNA (native tRNA^(Trp), G24A tRNA^(Trp) or the miscoding A9C tRNA^(Trp)) bound to cognate UGG or near-cognate UGA codons, determined at 3.2-Å resolution. The A9C and G24A mutations lead to miscoding by facilitating the distortion of tRNA required for decoding. A9C accomplishes this by increasing tRNA flexibility, whereas G24A allows the formation of an additional hydrogen bond that stabilizes the distortion. Our results also suggest that each native tRNA will adopt a unique conformation when delivered to the ribosome that allows accurate decoding.

Thomas A. Steitz, distinguished molecular and structural biologist, died on 9 October at the age of 78. Tom was known for his unerring judgment in picking fundamentally important problems, and persisting, sometimes for over a decade, until he solved them. His work on the information flow from DNA to RNA to protein culminated in work on the structure of the 50S subunit of the ribosome, for which he shared the 2009 Nobel Prize in Chemistry. Born on 23 August 1940 in Milwaukee, Wisconsin, Tom attended Lawrence College in Wisconsin on a scholarship and earned his degree in chemistry in 1962. He then began his graduate work at Harvard University. Although Tom initially planned to work on biophysical studies of nucleic acids, he was thrilled by lectures given by Max Perutz, the pioneer of protein crystallography, in which Max presented the first atomic-resolution structure of the protein myoglobin. Inspired to become a protein crystallographer, Tom joined the group of chemist and crystallographer W. N. (Bill) Lipscomb to work on the structure and mechanism of carboxypeptidase A. During this time, he met and married Joan Argetsinger Steitz, who became an equally distinguished molecular biologist. In 1966, Tom earned his Ph.D. in biochemistry and molecular biology at Harvard. The next year, he and Joan moved to the Medical Research Council (MRC) Laboratory of Molecular Biology (LMB) in Cambridge, United Kingdom, as postdocs, where Tom worked with crystallographer and biophysicist David Blow on the mechanism of protein hydrolysis by the serine protease chymotrypsin. The work led to an understanding of how all serine proteases cleave peptide bonds, as well as how the homologous trypsin recognizes positively charged side chains using a negatively charged aspartic acid at the bottom of its specificity pocket. Tom enjoyed discussions about research strategies with Brian Hartley and other pioneers of molecular and structural biology at LMB. He described his time at LMB as “the most important time in the development of my future research direction and my perspective on how creative science should best be done.” Next, Tom and Joan moved briefly to the University of California, Berkeley, where Tom had been offered a faculty job. As Tom described it, “I asked if there was any possibility of a job for Joan. The department chairman… said, ‘She's a woman. Women do not run their own lab; they work in the lab of their husband.’” So they departed for Yale University, where Fred Richards offered both Tom and Joan faculty positions as part of his visionary transformation of the Molecular Biophysics and Biochemistry Department into a powerhouse of structural and molecular biology. Joan said recently, “I was very, very lucky to have married Tom. He really believed that I should have as equal an opportunity to succeed as he.” They were a star couple at Yale, both becoming hugely influential leaders in their respective and sometimes overlapping fields. They even published an important paper together on the role of metal ions in catalysis by DNA and RNA polymerases. ![Figure][1] PHOTO: YALE UNIVERSITY/UPI/NEWSCOM At Yale, Tom began by determining the structure of hexokinase, an enzyme that phosphorylates glucose. The enzyme was one of the early examples of induced fit, whereby the substrate induces a change in enzyme conformation to bring catalytic residues into their active positions. His lab then focused on the structural mechanisms underlying the central dogma of molecular biology: how information flows from DNA to RNA to protein. His group worked out the structures of a series of important enzymes and cofactors that form the central core of structural molecular biology. This work culminated in his collaboration with Peter Moore, whose lifetime of work on the ribosome complemented Tom's crystallographic expertise. Together, they and their colleagues worked out the structure of the 50S ribosomal subunit, which definitively established that the ribosome is a ribozyme, and obtained the structures of complexes of the ribosome with many different antibiotics. In his steadfast determination to get to the heart of a problem, Tom was not afraid to make bold guesses about how a molecule works. This occasionally led to dramatically incorrect proposals. When he solved one of the first structures of a DNA-binding protein, the CAP repressor, he suggested that it bound to left-handed DNA. He joked that this was such an egregious mistake that he would have to solve five more structures before he would be invited to speak at a conference again. Later, he suggested a mechanism of peptidyl transfer in the 50 S ribosomal subunit. Both of these ideas proved incorrect. In each case, Tom exemplified how science should progress from mistakes by admitting the error and correcting it. His lab went on to show that CAP bound to a sharply bent form of DNA, a tour de force at the time. His lab also obtained a series of structures of substrate and transition-state analogs bound to the 50 S subunit that helped to establish the correct mechanism and was described by the Nobel Committee as the “jewel in the crown” of studies on the mechanism of peptidyl transfer. Rather than be overly wedded to his own ideas, Tom received corrections from others with interest as well. His ultimate goal was always to help establish the truth. Tom was known for his direct manner, which, along with his enviable track record, could intimidate anyone who was insecure. However, this was simply another aspect of his eagerness to get to the crux of the matter without wasting time. It also meant that he could be bluntly critical, often with an amusing pun, when encountering sloppy science, but generous with praise when he came across beautiful work. His students and colleagues never doubted where they stood with him. Even so, he was a supportive and egalitarian mentor who believed that talent and drive should determine success. These traits, along with his commitment to working only on problems he thought were truly important, helped him to train many generations of outstanding proteges, who along with achievements from his own lab form part of the huge legacy he has left behind. [1]: pending:yes

All organisms incorporate post-transcriptional modifications into ribosomal RNA, influencing ribosome assembly and function in ways that are poorly understood. The most highly conserved modification is the dimethylation of two adenosines near the 3′ end of the small subunit rRNA. Lack of these methylations due to deficiency in the KsgA methyltransferase stimulates translational errors during both the initiation and elongation phases of protein synthesis and confers resistance to the antibiotic kasugamycin. Here, we present the X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit lacking these dimethylations. Our data indicate that the KsgA-directed methylations facilitate structural rearrangements in order to establish a functionally optimum subunit conformation during the final stages of ribosome assembly.

The toxin colicin E3 targets the 30S subunit of bacterial ribosomes and cleaves a phosphodiester bond in the decoding center. We present the crystal structure of the 70S ribosome in complex with the cytotoxic domain of colicin E3 (E3-rRNase). The structure reveals how the rRNase domain of colicin binds to the A site of the decoding center in the 70S ribosome and cleaves the 16S ribosomal RNA (rRNA) between A1493 and G1494. The cleavage mechanism involves the concerted action of conserved residues Glu62 and His58 of the cytotoxic domain of colicin E3. These residues activate the 16S rRNA for 2' OH-induced hydrolysis. Conformational changes observed for E3-rRNase, 16S rRNA and helix 69 of 23S rRNA suggest that a dynamic binding platform is required for colicin E3 binding and function.

We report the crystal structure of release factor 2 bound to ribosome with an aminoacyl tRNA substrate analog at the ribosomal P site, at 3.1 Å resolution. The structure shows that upon stop-codon recognition, the universally conserved GGQ motif packs tightly into the peptidyl transferase center. Nucleotide A2602 of 23S rRNA, implicated in peptide release, packs with the GGQ motif in release factor 2. The ribose of A76 of the peptidyl-tRNA adopts the C2′-endo conformation, and the 2′ hydroxyl of A76 is within hydrogen-bond distance of the 2′ hydroxyl of A2451. The structure suggests how a catalytic water can be coordinated in the peptidyl transferase center and, together with previous biochemical and computational data, suggests a model for how the ester bond between the peptidyl tRNA and the nascent peptide is hydrolyzed.

Cell 139, 1084-1095 (2009). doi:10.1016/j.cell.2009.11.015, Published by Cell Press, [Cambridge, Mass.]

The high-resolution structures of ribosomal subunits published in 2000 have revolutionized the field of protein translation. They facilitated the determination and interpretation of functional complexes of the ribosome by crystallography and electron microscopy. Knowledge of the precise positions of residues in the ribosome in various states has facilitated increasingly sophisticated biochemical and genetic experiments, as well as the use of new methods such as single-molecule kinetics. In this review, we discuss how the interaction between structural and functional studies over the last decade has led to a deeper understanding of the complex mechanisms underlying translation.

The determination of the high-resolution structures of ribosomal subunits in the year 2000 and of the entire ribosome a few years later are revolutionizing our understanding of the role of the ribosome in translation. In the present article, I summarize the main contributions from our laboratory to this worldwide effort. These include the determination of the structure of the 30S ribosomal subunit and its complexes with antibiotics, the role of the 30S subunit in decoding, and the high-resolution structure of the entire 70S ribosome complexed with mRNA and tRNA.

Post-transcriptional modifications at the first (wobble) position of the tRNA anticodon participate in precise decoding of the genetic code. To decode codons that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine derivatives (xm(5)U) at the wobble position. However, the functional properties of the C5-substituents of xm(5)U in codon recognition remain elusive. We previously found that mitochondrial tRNAs(Leu(UUR)) with pathogenic point mutations isolated from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine (taum(5)U) modification and caused a decoding defect. Here, we constructed Escherichia coli tRNAs(Leu(UUR)) with or without xm(5)U modifications at the wobble position and measured their decoding activities in an in vitro translation as well as by A-site tRNA binding. In addition, the decoding properties of tRNA(Arg) lacking mnm(5)U modification in a knock-out strain of the modifying enzyme (DeltamnmE) were examined by pulse labeling using reporter constructs with consecutive AGR codons. Our results demonstrate that the xm(5)U modification plays a critical role in decoding NNG codons by stabilizing U.G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing taum(5)U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the taum(5)U.G base pair does not have classical U.G wobble geometry. These structures provide help to explain how the taum(5)U modification enables efficient decoding of UUG codons.

One of the most prevalent base modifications involved in decoding is uridine 5-oxyacetic acid at the wobble position of tRNA. It has been known for several decades that this modification enables a single tRNA to decode all four codons in a degenerate codon box. We have determined structures of an anticodon stem-loop of tRNA(Val) containing the modified uridine with all four valine codons in the decoding site of the 30S ribosomal subunit. An intramolecular hydrogen bond involving the modification helps to prestructure the anticodon loop. We found unusual base pairs with the three noncomplementary codon bases, including a G.U base pair in standard Watson-Crick geometry, which presumably involves an enol form for the uridine. These structures suggest how a modification in the uridine at the wobble position can expand the decoding capability of a tRNA.

Summary Initiation of translation is the process by which initiator tRNA and the start codon of mRNA are positioned in the ribosomal P site. In eukaryotes, one of the first steps involves the binding of two small factors, eIF1 and eIF1A, to the small (40S) ribosomal subunit. This facilitates tRNA binding, allows scanning of mRNA, and maintains fidelity of start codon recognition. Using cryo-EM, we have obtained 3D reconstructions of 40S bound to both eIF1 and eIF1A, and with each factor alone. These structures reveal that together, eIF1 and eIF1A stabilize a conformational change that opens the mRNA binding channel. Biochemical data reveal that both factors accelerate the rate of ternary complex (eIF2•GTP•Met-tRNA i Met ) binding to 40S but only eIF1A stabilizes this interaction. Our results suggest that eIF1 and eIF1A promote an open, scanning-competent preinitiation complex that closes upon start codon recognition and eIF1 release to stabilize ternary complex binding and clamp down on mRNA.

Termination of protein synthesis occurs when a translating ribosome encounters one of three universally conserved stop codons: UAA, UAG or UGA. Release factors recognize stop codons in the ribosomal A-site to mediate release of the nascent chain and recycling of the ribosome. Bacteria decode stop codons using two separate release factors with differing specificities for the second and third bases. By contrast, eukaryotes rely on an evolutionarily unrelated omnipotent release factor (eRF1) to recognize all three stop codons. The molecular basis of eRF1 discrimination for stop codons over sense codons is not known. Here we present cryo-electron microscopy (cryo-EM) structures at 3.5-3.8 A resolution of mammalian ribosomal complexes containing eRF1 interacting with each of the three stop codons in the A-site. Binding of eRF1 flips nucleotide A1825 of 18S ribosomal RNA so that it stacks on the second and third stop codon bases. This configuration pulls the fourth position base into the A-site, where it is stabilized by stacking against G626 of 18S rRNA. Thus, eRF1 exploits two rRNA nucleotides also used during transfer RNA selection to drive messenger RNA compaction. In this compacted mRNA conformation, stop codons are favoured by a hydrogen-bonding network formed between rRNA and essential eRF1 residues that constrains the identity of the bases. These results provide a molecular framework for eukaryotic stop codon recognition and have implications for future studies on the mechanisms of canonical and premature translation termination.

During protein synthesis, translational release factors catalyze the release of the polypeptide chain when a stop codon on the mRNA reaches the A site of the ribosome. The detailed mechanism of this process is currently unknown. We present here the crystal structures of the ribosome from Thermus thermophilus with RF1 and RF2 bound to their cognate stop codons, at resolutions of 5.9 Angstrom and 6.7 Angstrom, respectively. The structures reveal details of interactions of the factors with the ribosome and mRNA, including elements previously implicated in decoding and peptide release. They also shed light on conformational changes both in the factors and in the ribosome during termination. Differences seen in the interaction of RF1 and RF2 with the L11 region of the ribosome allow us to rationalize previous biochemical data. Finally, this work demonstrates the feasibility of crystallizing ribosomes with bound factors at a defined state along the translational pathway.

The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNALysUUU requires the modification N6-threonylcarbamoyladenosine at position 37 (t6A37), adjacent and 3′ to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t6A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm5U34 or s2U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.