Project description:The structural understanding of eukaryotic translation lags behind that of translation on bacterial ribosomes. Here, we present two subnanometer resolution structures of S. cerevisiae 80S ribosome complexes formed with either one or two tRNAs and bound in response to an mRNA fragment containing the Kozak consensus sequence. The ribosomes adopt two globally different conformations that are related to each other by the rotation of the small subunit. Comparison with bacterial ribosome complexes reveals that the global structures and modes of intersubunit rotation of the yeast ribosome differ significantly from those in the bacterial counterpart, most notably in the regions involving the tRNA, small ribosomal subunit, and conserved helix 69 of the large ribosomal subunit. The structures provide insight into ribosome dynamics implicated in tRNA translocation and help elucidate the role of the Kozak fragment in positioning an open reading frame during translation initiation in eukaryotes.
Project description:Upf1, Upf2, and Upf3 are the central regulators of nonsense-mediated mRNA decay (NMD), the eukaryotic mRNA quality control pathway generally triggered when a premature termination codon is recognized by the ribosome. The NMD-related functions of the Upf proteins likely commence while these factors are ribosome-associated, but little is known of the timing of their ribosome binding, their specificity for ribosomes translating NMD substrates, or the nature and role of any ribosome:Upf complexes. Here, we have elucidated details of the ribosome-associated steps of NMD. By combining yeast genetics with selective ribosome profiling and co-sedimentation analyses of polysomes with wild-type and mutant Upf proteins, our approaches have identified distinct states of ribosome:Upf association. All three Upf factors manifest progressive polysome association as mRNA translation proceeds, but these events appear to be preceded by formation of a Upf1:80S complex as mRNAs initiate translation. This complex is likely executing an early mRNA surveillance function.
Project description:Translocation moves the tRNA2?mRNA module directionally through the ribosome during the elongation phase of protein synthesis. Although translocation is known to entail large conformational changes within both the ribosome and tRNA substrates, the orchestrated events that ensure the speed and fidelity of this critical aspect of the protein synthesis mechanism have not been fully elucidated. Here, we present three high-resolution structures of intermediates of translocation on the mammalian ribosome where, in contrast to bacteria, ribosomal complexes containing the translocase eEF2 and the complete tRNA2?mRNA module are trapped by the non-hydrolyzable GTP analog GMPPNP. Consistent with the observed structures, single-molecule imaging revealed that GTP hydrolysis principally facilitates rate-limiting, final steps of translocation, which are required for factor dissociation and which are differentially regulated in bacterial and mammalian systems by the rates of deacyl-tRNA dissociation from the E site.
Project description:An 11.7-A-resolution cryo-EM map of the yeast 80S.eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcin-ricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet-like subunit rearrangement (RSR) occurs in the 80S.eEF2.sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction.
Project description:Upf1, Upf2, and Upf3 are the central regulators of nonsense-mediated mRNA decay (NMD), the eukaryotic mRNA quality control pathway generally triggered when a premature termination codon is recognized by the ribosome. The NMD-related functions of the Upf proteins likely commence while these factors are ribosome-associated, but little is known of the timing of their ribosome binding, their specificity for ribosomes translating NMD substrates, or the nature and role of any ribosome:Upf complexes. Here, we have elucidated details of the ribosome-associated steps of NMD. By combining yeast genetics with selective ribosome profiling and co-sedimentation analyses of polysomes with wild-type and mutant Upf proteins, our approaches have identified distinct states of ribosome:Upf association. All three Upf factors manifest progressive polysome association as mRNA translation proceeds, but these events appear to be preceded by formation of a Upf1:80S complex as mRNAs initiate translation. This complex is likely executing an early mRNA surveillance function.
Project description:On the basis of kinetic data on ribosome protein synthesis, the mechanical energy for translocation of the mRNA-tRNA complex is thought to be provided by GTP hydrolysis of an elongation factor (eEF2 in eukaryotes, EF-G in bacteria). We have obtained cryo-EM reconstructions of eukaryotic ribosomes complexed with ADP-ribosylated eEF2 (ADPR-eEF2), before and after GTP hydrolysis, providing a structural basis for analyzing the GTPase-coupled mechanism of translocation. Using the ADP-ribosyl group as a distinct marker, we observe conformational changes of ADPR-eEF2 that are due strictly to GTP hydrolysis. These movements are likely representative of native eEF2 motions in a physiological context and are sufficient to uncouple the mRNA-tRNA complex from two universally conserved bases in the ribosomal decoding center (A1492 and A1493 in Escherichia coli) during translocation. Interpretation of these data provides a detailed two-step model of translocation that begins with the eEF2/EF-G binding-induced ratcheting motion of the small ribosomal subunit. GTP hydrolysis then uncouples the mRNA-tRNA complex from the decoding center so translocation of the mRNA-tRNA moiety may be completed by a head rotation of the small subunit.
Project description:Methodology was developed for specifically anchoring Escherichia coli 70S ribosomes onto a chemically modified, cysteine-reactive glass surface. Immobilized ribosomes maintain the capability of binding a polyuridylic acid (poly(U)) template, enabling investigation of mechanical properties of individual ribosome-poly(U) complexes using laser tweezers. Streptavidin-coated polystyrene microspheres bound specifically to the biotinylated 3' end of long (up to 10,000 bases) poly(U) strands. A novel optical method was built to control the position of the laser trap along the microscope optical axis at 2 nm resolution, facilitating measurement of the force-extension relationship for poly(U). Some immobilized ribosome-poly(U) complexes supported 100 pN of force applied at the 3' end of the mRNA. Binding of N-acetylated Phe-tRNA(Phe), an analog of the initiator fMet-tRNA(Met), enhanced the population of complexes that could withstand high forces. The persistence length of poly(U) RNA homopolymer, modeled as a worm-like chain, was found to be 0.79 +/- 0.05 nm and the backbone elasticity was 900 +/- 140 pN, similar to values for single-stranded DNA.
Project description:Protein synthesis requires the accurate positioning of mRNA and tRNA in the peptidyl-tRNA site of the ribosome. Here we describe x-ray crystal structures of the intact bacterial ribosome from Escherichia coli in a complex with mRNA and the anticodon stem-loop of P-site tRNA. At 3.5-A resolution, these structures reveal rearrangements in the intact ribosome that clamp P-site tRNA and mRNA on the small ribosomal subunit. Binding of the anticodon stem-loop of P-site tRNA to the ribosome is sufficient to lock the head of the small ribosomal subunit in a single conformation, thereby preventing movement of mRNA and tRNA before mRNA decoding.
Project description:We have carried out molecular dynamics simulations of the tRNA anticodon and mRNA codon, inside the ribosome, to study the effect of the common tRNA modifications cmo(5)U34 and m(6)A37. In tRNA(Val), these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. Previous data suggest that entropic effects are mainly responsible for the extended reading capabilities, but detailed mechanisms have remained unknown. We have performed a wide range of simulations to elucidate the details of these mechanisms at the atomic level and quantify their effects: extensive free energy perturbation coupled with umbrella sampling, entropy calculations of tRNA (free and bound to the ribosome), and thorough structural analysis of the ribosomal decoding center. No prestructuring effect on the tRNA anticodon stem-loop from the two modifications could be observed, but we identified two mechanisms that may contribute to the expanded decoding capability by the modifications: The further reach of the cmo(5)U34 allows an alternative outer conformation to be formed for the noncognate base pairs, and the modification results in increased contacts between tRNA, mRNA, and the ribosome.