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 joining of ribosomal subunits in eukaryotes requires eIF5B

Initiation of eukaryotic protein synthesis begins with the ribosome separated into its 40S and 60S subunits. The 40S subunit first binds eukaryotic initiation factor (eIF) 3 and an eIF2-GTP-initiator transfer RNA ternary complex. The resulting complex requires eIF1, eIF1A, eIF4A, eIF4B and eIF4F to bind to a messenger RNA and to scan to the initiation codon. eIF5 stimulates hydrolysis of eIF2-bound GTP and eIF2 is released from the 48S complex formed at the initiation codon before it is joined by a 60S subunit to form an active 80S ribosome. Here we show that hydrolysis of eIF2-bound GTP induced by eIF5 in 48S complexes is necessary but not sufficient for the subunits to join. A second factor termed eIF5B (relative molecular mass 175,000) is essential for this process. It is a homologue of the prokaryotic initiation factor IF2 (re and, like it, mediates joining of subunits and has a ribosome-dependent GTPase activity that is essential for its function.     

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?? 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.     

Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA

The elongation factors EF-Tu and EF-G interact with ribosomes during protein synthesis: EF-Tu presents incoming aminoacyl transfer RNA to the programmed ribosome as an EF-Tu-GTP-tRNA ternary complex and EF-G promotes translocation of peptidyl-tRNA and its associated messenger RNA from the A to the P site after peptidyl transfer. Both events are accompanied by ribosome-dependent GTP hydrolysis. Here we use chemical probes to investigate the possible interaction of these factors with ribosomal RNA in E. coli ribosomes. We observe EF-G-dependent footprints in vitro and in vivo around position 1,067 in domain II of 23S rRNA, and in the loop around position 2,660 in domain VI.EF-Tu gives an overlapping footprint in vitro at positions 2,655 and 2,661, but shows no effect at position 1,067. The 1,067 region is the site of interaction of the antibiotic thiostrepton, which prevents formation of the EF-G-GTP-ribosome complex and is a site for interaction with the GTPase-related protein L11 (ref. 3). The universally conserved loop in the 2,660 region is the site of attack by the RNA-directed cytotoxins alpha-sarcin and ricin, whose effects abolish translation and include the loss of elongation factor-dependent functions in eukaryotic ribosomes.     

The complex of tmRNA-SmpB and EF-G on translocating ribosomes

Bacterial ribosomes stalled at the 3' end of malfunctioning messenger RNAs can be rescued by transfer-messenger RNA (tmRNA)-mediated trans-translation. The SmpB protein forms a complex with the tmRNA, and the transfer-RNA-like domain (TLD) of the tmRNA then enters the A site of the ribosome. Subsequently, the TLD-SmpB module is translocated to the P site, a process that is facilitated by the elongation factor EF-G, and translation is switched to the mRNA-like domain (MLD) of the tmRNA. Accurate loading of the MLD into the mRNA path is an unusual initiation mechanism. Despite various snapshots of different ribosome-tmRNA complexes at low to intermediate resolution, it is unclear how the large, highly structured tmRNA is translocated and how the MLD is loaded. Here we present a cryo-electron microscopy reconstruction of a fusidic-acid-stalled ribosomal 70S-tmRNA-SmpB-EF-G complex (carrying both of the large ligands, that is, EF-G and tmRNA) at 8.3?? resolution. This post-translocational intermediate (TI(POST)) presents the TLD-SmpB module in an intrasubunit ap/P hybrid site and a tRNA(fMet) in an intrasubunit pe/E hybrid site. Conformational changes in the ribosome and tmRNA occur in the intersubunit space and on the solvent side. The key underlying event is a unique extra-large swivel movement of the 30S head, which is crucial for both tmRNA-SmpB translocation and MLD loading, thereby coupling translocation to MLD loading. This mechanism exemplifies the versatile, dynamic nature of the ribosome, and it shows that the conformational modes of the ribosome that normally drive canonical translation can also be used in a modified form to facilitate more complex tasks in specialized non-canonical pathways.     

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.     

Visualization of release factor 3 on the ribosome during termination of protein synthesis

Termination of protein synthesis by the ribosome requires two release factor (RF) classes. The class II RF3 is a GTPase that removes class I RFs (RF1 or RF2) from the ribosome after release of the nascent polypeptide. RF3 in the GDP state binds to the ribosomal class I RF complex, followed by an exchange of GDP for GTP and release of the class I RF. As GTP hydrolysis triggers release of RF3 (ref. 4), we trapped RF3 on Escherichia coli ribosomes using a nonhydrolysable GTP analogue. Here we show by cryo-electron microscopy that the complex can adopt two different conformational states. In 'state 1', RF3 is pre-bound to the ribosome, whereas in 'state 2' RF3 contacts the ribosome GTPase centre. The transfer RNA molecule translocates from the peptidyl site in state 1 to the exit site in state 2. This translocation is associated with a large conformational rearrangement of the ribosome. Because state 1 seems able to accommodate simultaneously both RF3 and RF2, whose position is known from previous studies, we can infer the release mechanism of class I RFs.     

Structure of the 30S translation initiation complex

Translation initiation, the rate-limiting step of the universal process of protein synthesis, proceeds through sequential, tightly regulated steps. In bacteria, the correct messenger RNA start site and the reading frame are selected when, with the help of initiation factors IF1, IF2 and IF3, the initiation codon is decoded in the peptidyl site of the 30S ribosomal subunit by the fMet-tRNA(fMet) anticodon. This yields a 30S initiation complex (30SIC) that is an intermediate in the formation of the 70S initiation complex (70SIC) that occurs on joining of the 50S ribosomal subunit to the 30SIC and release of the initiation factors. The localization of IF2 in the 30SIC has proved to be difficult so far using biochemical approaches, but could now be addressed using cryo-electron microscopy and advanced particle separation techniques on the basis of three-dimensional statistical analysis. Here we report the direct visualization of a 30SIC containing mRNA, fMet-tRNA(fMet) and initiation factors IF1 and GTP-bound IF2. We demonstrate that the fMet-tRNA(fMet) is held in a characteristic and precise position and conformation by two interactions that contribute to the formation of a stable complex: one involves the transfer RNA decoding stem which is buried in the 30S peptidyl site, and the other occurs between the carboxy-terminal domain of IF2 and the tRNA acceptor end. The structure provides insights into the mechanism of 70SIC assembly and rationalizes the rapid activation of GTP hydrolysis triggered on 30SIC-50S joining by showing that the GTP-binding domain of IF2 would directly face the GTPase-activated centre of the 50S subunit.     

Heterogeneous pathways and timing of factor departure during translation initiation

The initiation of translation establishes the reading frame for protein synthesis and is a key point of regulation. Initiation involves factor-driven assembly at a start codon of a messenger RNA of an elongation-competent 70S ribosomal particle (in bacteria) from separated 30S and 50S subunits and initiator transfer RNA. Here we establish in Escherichia coli, using direct single-molecule tracking, the timing of initiator tRNA, initiation factor 2 (IF2; encoded by infB) and 50S subunit joining during initiation. Our results show multiple pathways to initiation, with orders of arrival of tRNA and IF2 dependent on factor concentration and composition. IF2 accelerates 50S subunit joining and stabilizes the assembled 70S complex. Transition to elongation is gated by the departure of IF2 after GTP hydrolysis, allowing efficient arrival of elongator tRNAs to the second codon presented in the aminoacyl-tRNA binding site (A site). These experiments highlight the power of single-molecule approaches to delineate mechanisms in complex multicomponent systems.     

The transorientation hypothesis for codon recognition during protein synthesis

During decoding, a codon of messenger RNA is matched with its cognate aminoacyl-transfer RNA and the amino acid carried by the tRNA is added to the growing protein chain. Here we propose a molecular mechanism for the decoding phase of translation: the transorientation hypothesis. The model incorporates a newly identified tRNA binding site and utilizes a flip between two tRNA anticodon loop structures, the 5'-stacked and the 3'-stacked conformations. The anticodon loop acts as a three-dimensional hinge permitting rotation of the tRNA about a relatively fixed codon-anticodon pair. This rotation, driven by a conformational change in elongation factor Tu involving GTP hydrolysis, transorients the incoming tRNA into the A site from the D site of initial binding and decoding, where it can be proofread and accommodated. The proposed mechanisms are compatible with the known structures, conformations and functions of the ribosome and its component parts including tRNAs and EF-Tu, in both the GTP and GDP states.     

Eukaryotic initiation factor 6 is rate-limiting in translation, growth and transformation

Cell growth and proliferation require coordinated ribosomal biogenesis and translation. Eukaryotic initiation factors (eIFs) control translation at the rate-limiting step of initiation. So far, only two eIFs connect extracellular stimuli to global translation rates: eIF4E acts in the eIF4F complex and regulates binding of capped messenger RNA to 40S subunits, downstream of growth factors, and eIF2 controls loading of the ternary complex on the 40S subunit and is inhibited on stress stimuli. No eIFs have been found to link extracellular stimuli to the activity of the large 60S ribosomal subunit. eIF6 binds 60S ribosomes precluding ribosome joining in vitro. However, studies in yeasts showed that eIF6 is required for ribosome biogenesis rather than translation. Here we show that mammalian eIF6 is required for efficient initiation of translation, in vivo. eIF6 null embryos are lethal at preimplantation. Heterozygous mice have 50% reduction of eIF6 levels in all tissues, and show reduced mass of hepatic and adipose tissues due to a lower number of cells and to impaired G1/S cell cycle progression. eIF6(+/-) cells retain sufficient nucleolar eIF6 and normal ribosome biogenesis. The liver of eIF6(+/-) mice displays an increase of 80S in polysomal profiles, indicating a defect in initiation of translation. Consistently, isolated hepatocytes have impaired insulin-stimulated translation. Heterozygous mouse embryonic fibroblasts recapitulate the organism phenotype and have normal ribosome biogenesis, reduced insulin-stimulated translation, and delayed G1/S phase progression. Furthermore, eIF6(+/-) cells are resistant to oncogene-induced transformation. Thus, eIF6 is the first eIF associated with the large 60S subunit that regulates translation in response to extracellular signals.     

Internal initiation of translation mediated by the 5' leader of a cellular mRNA.

A Robosome-scanning model has been proposed to explain the initiation of eukaryotic messenger RNAs in which binding of the 43S ternary ribosomal subunit near or at the 5' end of the mRNA is facilitated by an interaction between the methylated cap-structure at the end of the mRNA and the cap-binding protein complex eIF-4F. But picornaviral mRNAs do not have a 5' terminal cap structure and are translated by internal ribosome binding. A cellular mRNA, encoding the immunoglobulin heavy-chain binding protein, can be translated in poliovirus-infected cells at a time when cap-dependent translation of host cell mRNAs is inhibited. We report here that the 5' leader of the binding protein mRNA can directly confer internal ribosome binding to an mRNA in mammalian cells, indicating that translation initiation by an internal ribosome-binding mechanism is used by eukaryotic mRNAs.     

Structure of the signal recognition particle interacting with the elongation-arrested ribosome

Cotranslational translocation of proteins across or into membranes is a vital process in all kingdoms of life. It requires that the translating ribosome be targeted to the membrane by the signal recognition particle (SRP), an evolutionarily conserved ribonucleoprotein particle. SRP recognizes signal sequences of nascent protein chains emerging from the ribosome. Subsequent binding of SRP leads to a pause in peptide elongation and to the ribosome docking to the membrane-bound SRP receptor. Here we present the structure of a targeting complex consisting of mammalian SRP bound to an active 80S ribosome carrying a signal sequence. This structure, solved to 12 A by cryo-electron microscopy, enables us to generate a molecular model of SRP in its functional conformation. The model shows how the S domain of SRP contacts the large ribosomal subunit at the nascent chain exit site to bind the signal sequence, and that the Alu domain reaches into the elongation-factor-binding site of the ribosome, explaining its elongation arrest activity.     

Substrate twinning activates the signal recognition particle and its receptor

Signal sequences target proteins for secretion from cells or for integration into cell membranes. As nascent proteins emerge from the ribosome, signal sequences are recognized by the signal recognition particle (SRP), which subsequently associates with its receptor (SR). In this complex, the SRP and SR stimulate each other's GTPase activity, and GTP hydrolysis ensures unidirectional targeting of cargo through a translocation pore in the membrane. To define the mechanism of reciprocal activation, we determined the 1.9 A structure of the complex formed between these two GTPases. The two partners form a quasi-two-fold symmetrical heterodimer. Biochemical analysis supports the importance of the extensive interaction surface. Complex formation aligns the two GTP molecules in a symmetrical, composite active site, and the 3'OH groups are essential for association, reciprocal activation and catalysis. This unique circle of twinned interactions is severed twice on hydrolysis, leading to complex dissociation after cargo delivery.     

Subnanometre-resolution structure of the intact Thermus thermophilus H+-driven ATP synthase

Ion-translocating rotary ATPases serve either as ATP synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. During ATP synthesis, ion translocation through the membrane-bound region of the complex causes rotation of a central rotor that drives conformational changes and ATP synthesis in the catalytic region of the complex. There are no structural models available for the intact membrane region of any ion-translocating rotary ATPase. Here we present a 9.7?? resolution map of the H(+)-driven ATP synthase from Thermus thermophilus obtained by electron cryomicroscopy of single particles in ice. The 600-kilodalton complex has an overall subunit composition of A(3)B(3)CDE(2)FG(2)IL(12). The membrane-bound motor consists of a ring of L subunits and the carboxy-terminal region of subunit I, which are equivalent to the c and a subunits of most other rotary ATPases, respectively. The map shows that the ring contains 12 L subunits and that the I subunit has eight transmembrane helices. The L(12) ring and I subunit have a surprisingly small contact area in the middle of the membrane, with helices from the I subunit making contacts with two different L subunits. The transmembrane helices of subunit I form bundles that could serve as half-channels across the membrane, with the first half-channel conducting protons from the periplasm to the L(12) ring and the second half-channel conducting protons from the L(12) ring to the cytoplasm. This structure therefore suggests the mechanism by which a transmembrane proton motive force is converted to rotation in rotary ATPases.     

Two-step binding of eukaryotic ribosomes to brome mosaic virus RNA3

Although brome mosaic virus RNA3 has only one translatable cistron, it can bind two 80S ribosomes at initiation. One ribosome binds at the first AUG codon (base 92-94). The other binds nearer the 5' end at an entry or holding site. Disome formation is thus unrelated to a silent cistron approximately 1,000 bases downstream.     

Structure of the 30S ribosomal subunit

Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.     

Subsecond deactivation of transducin by endogenous GTP hydrolysis

The response of a retinal rod cell to a weak flash of light is mediated by a receptor/GTP-binding protein (rhodopsin/transducin) signal transduction system and terminates within a second. The T alpha subunit of transducin (composed of subunits T alpha, T beta and T gamma) is triggered by photoexcited rhodopsin (R*) to release GDP and bind GTP. The binding of GTP causes release of the T alpha unit from T beta gamma and allows it to modulate the activity of an enzyme that generates a second messenger. Termination of the response requires the hydrolysis of the GTP by intrinsic GTPase. As with other G proteins, the GTPase activity of transducin seems to be slow. Reported in vitro turnover rates of a few molecules of GTP hydrolysed per molecule of transducin per minute imply a T alpha-GTP deactivation time of many seconds. But this time might be only a small fraction of that of the GTPase cycle. We have now used time-resolved microcalorimetry in bovine rod outer segments (ROS) to monitor the heat release due to the hydrolysis of GTP by a transducin population that had been quickly activated by flash illumination of rhodopsin. The enthalpy of GTP hydrolysis is released within 1 s at 23 degrees C. This deactivation time seems to be independent of any diffusible factor in the preparation and concurs with the termination kinetics of the rod's response. Thereafter, transducin seems unable to reload GTP for many seconds. This refractory 'resetting' time may account for the low steady-state GTPase rates in vitro.     

Peptide bond formation destabilizes Shine-Dalgarno interaction on the ribosome

The ribosome is a molecular machine that translates the genetic code contained in the messenger RNA into an amino acid sequence through repetitive cycles of transfer RNA selection, peptide bond formation and translocation. Here we demonstrate an optical tweezer assay to measure the rupture force between a single ribosome complex and mRNA. The rupture force was compared between ribosome complexes assembled on an mRNA with and without a strong Shine-Dalgarno (SD) sequence-a sequence found just upstream of the coding region of bacterial mRNAs, involved in translation initiation. The removal of the SD sequence significantly reduced the rupture force in complexes carrying an aminoacyl tRNA, Phe-tRNA(Phe), in the A site, indicating that the SD interactions contribute significantly to the stability of the ribosomal complex on the mRNA before peptide bond formation. In contrast, the presence of a peptidyl tRNA analogue, N-acetyl-Phe-tRNA(Phe), in the A site, which mimicked the post-peptidyl transfer state, weakened the rupture force as compared to the complex with Phe-tRNA(Phe), and the resultant force was the same for both the SD-containing and SD-deficient mRNAs. These results suggest that formation of the first peptide bond destabilizes the SD interaction, resulting in the weakening of the force with which the ribosome grips an mRNA. This might be an important requirement to facilitate movement of the ribosome along mRNA during the first translocation step.     

Crystal structure of metarhodopsin II

G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular heterotrimeric G proteins (Gαβγ). The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta?II) is formed, in which the retinal Schiff base is still intact but deprotonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplasmic crevice for uptake of the interacting C terminus of the Gα subunit. Owing to Schiff base hydrolysis, Meta?II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta?II, presuming that the crystal's high concentration of opsin in an active conformation (Ops*) may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0?? and 2.85?? crystal structures, respectively, of Meta?II alone or in complex with an 11-amino-acid C-terminal fragment derived from Gα (GαCT2). GαCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Gα-derived peptide to Ops* (ref. 7). In the Meta?II structures, the electron density from the retinal ligand seamlessly continues into the Lys?296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all-trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta?II. The structures can now serve as models for the large GPCR family.