tRNA tKUUU,tQUUG, and tEUUC wobble position modifications fine-tune protein translation by promoting ribosome A-site binding

tRNA modifications are crucial to ensure translation efficiency and fidelity. In eukaryotes, the URM1 and ELP pathways increase cellular resistance to various stress conditions, such as nutrient starvation and oxidative agents, by promoting thiolation and methoxycarbonylmethylation, respectively, of the wobble uridine of cytoplasmic (tK^UUU), (tQ^UUG), and (tE^UUC). Although in vitro experiments have implicated these tRNA modifications in modulating wobbling capacity and translation efficiency, their exact in vivo biological roles remain largely unexplored. Using a combination of quantitative proteomics and codon-specific translation reporters, we find that translation of a specific gene subset enriched for AAA, CAA, and GAA codons is impaired in the absence of URM1- and ELP-dependent tRNA modifications. Moreover, in vitro experiments using native tRNAs demonstrate that both modifications enhance binding of tK^UUU to the ribosomal A-site. Taken together, our data suggest that tRNA thiolation and methoxycarbonylmethylation regulate translation of genes with specific codon content.     

The nature of peptides presented by an HLA class I low expression allele

Background

The functional integrity of human leukocyte antigen low expression variants is a prerequisite for considering them as essential in the matching process of hematopoietic stem cell donors and recipients to diminish the risk of serious complications such as graft-versus-host disease or graft rejection. The HLA-A*3014L variant has a disulfide bridge missing in the α2 domain which could affect peptide binding and presentation to T cells.

Design and Methods

HLA-A*3014L and HLA-A*3001 were expressed as truncated variants and peptides were eluted and subjected to pool sequencing by Edman degradation as well as to single-peptide sequencing by mass spectrometry. Quantitative analysis of binding peptides presented in vivo was performed by a flow cytometric peptide-binding assay using HLA-A*3001 and HLA-A*3014L-expressing B-LCLs.

Results

The truncated HLA-A*3014L protein was secreted in the supernatant and it was possible to elute and sequence peptides. Sequence analysis of these eluted peptides revealed no relevant differences to the peptide motif of HLA-A*3001, indicating that the Cys164Ser substitution does not substantially alter the spectrum of presented peptides. Strong binding of one of the shared in vivo identified HLA-A*3001/3014L ligands was confirmed in the peptide-binding assay.

Conclusions

This study is the first to demonstrate that HLA low expression variants are able to present peptides and, thus, can be considered as functionally active. When comparing peptide motifs, it is likely that HLA-A*3014L and HLA-A*3001 represent a permissive mismatch with low allogenicity in hematopoietic stem cell transplantation. These results indicate that surface expression, as well as peptide-binding data of HLA variants with similar disulfide bridge variations (e.g. HLA-A*3211Q) need to be considered as functionally active in an allogeneic hematopoietic stem cell transplantation setting as long as the opposite has not been shown. Otherwise a relevant but not considered HLA mismatch could result in a severe allogeneic T-cell response and graft-versus-host disease.     

The role of fluctuations in tRNA selection by the ribosome

The detailed mechanism of how the ribosome decodes protein sequence information with an abnormally high accuracy, after 40 years of study, remains elusive. A critical element in selecting correct transfer RNA (tRNA) transferring correct amino acid is "induced fit" between the ribosome and tRNA. By using single-molecule methods, the induced fit mechanism is shown to position favorably the correct tRNA after initial codon recognition. We provide evidence that this difference in positioning and thermal fluctuations constitutes the primary mechanism for the initial selection of tRNA. This work demonstrates thermal fluctuations playing a critical role in the substrate selection by an enzyme.     

Self-reported overdose among injecting drug users in London: extent and nature of the problem

Aims. To estimate the extent and nature of overdose and factors associated with overdose among injecting drug users in London. Design. Three hundred and twelve current injecting drug users were recruited and interviewed in community settings by a team of "privileged access interviewers". Measurements. A structured questionnaire was used that covered the following areas: demographic characteristics, drug use, injecting behaviour, sharing practices, severity of drug dependence, experience of overdose, injecting-related health problems and treatment history. Findings. The results showed that experience of overdose was common (38%). A majority (54%) had witnessed someone else overdose. Overdosing was not a solitary experience; over 80% of subjects who had overdosed had done so in the presence of someone else, but only 27% reported ambulances having been called. Factors found to be associated with overdose were: age at which injecting began; gender (women being more likely to experience overdose); use of alcohol; and polydrug injection. The overall rate of overdosing was one per 6 years of injecting; however, once an individual had an overdose the chance of having another increased. The risk of experiencing a first overdose fell with years of injecting. Conclusions. Harm-reduction interventions with drug injectors should educate users on the risk factors associated with overdose and actions that should be taken when someone has overdosed. Interventions designed to reduce the risk of overdose may be more effective if they are differentially targeted on drug injectors who have already experienced an overdose.     

Quantification of soluble HLA class I gene products by an enzyme linked immunosorbent assay

Summary A simplified enzyme linked immunosorbent assay utilizing an HLA class I frameworkspecific monoclonal antibody and a polyclonal enzyme linked beta-2 microglobulin specific antiserum has been established for the quantitative measurement of soluble HLA class I molecules. A total of 219 unrelated healthy individuals and 137 members of 28 families typed for HLA were analyzed for their non-membrane bound, i.e. soluble HLA-A,B,C antigens (sHLA-A,B,C). As reported by others, we observed associations of higher or lower sHLA-A,B,C values to particular HLA antigens: High plasma values were observed in probands positive for HLA-A23, A24, A29, Aw33, Bw65, and Cw8 and low values in HLA-B27 and B37 positive individuals. However, as shown by family studies, levels of sHLA-A,B,C were apparently not controlled by the MHC haplotypes alone, since no significant difference between HLA identical siblings and two haplotype different individuals could be detected. Thus, additional non-MHC linked gene(s) may be involved in the release of class I gene products.Supported in part by a research grant from the Fresenius AG, Oberursel, Federal Republic of Germany     

The ribosome uses cooperative conformational changes to maximize and regulate the efficiency of translation

One of the most challenging unanswered questions regarding the structural biology of biomolecular machines such as the two-subunit ribosome is whether and how these machines coordinate seemingly independent and random conformational fluctuations to maximize and regulate their functional efficiencies. To address this question, we have used ribosome mutagenesis or a ribosome-targeting antibiotic to predictably perturb the dynamics of intersubunit rotation, a structural rearrangement of the ribosome that is essential for the translocation and ejection of ribosome-bound tRNAs during translation. Concomitantly, we have used single-molecule fluorescence resonance energy transfer (smFRET) to characterize the effects of these perturbations on the dynamics of ribosomal L1 stalk movements and ribosome-bound tRNA reconfigurations, conformational changes that are likewise essential for the translocation and ejection of tRNAs during translation. Together with the results of complementary biochemical studies, our smFRET studies demonstrate that the ribosome uses cooperative conformational changes to maximize and regulate the efficiency with which it translocates and ejects tRNAs during translation. We propose that the ribosome employs cooperative conformational changes to efficiently populate global conformational states that are productive for translation, that translation factors exploit this cooperativity as part of their mechanisms of action, and that antibiotics exploit it to maximize the potency with which they inhibit translation. It is likely that similar cooperative conformational changes underlie the function and regulation of other biomolecular machines.During the catalytic cycle of many enzymes, multiple, spatially distant enzyme structural elements must often undergo functionally important conformational changes (1). Consistent with the view that such structural rearrangements must be rapidly organized and executed to maintain catalytic efficiency, many recent studies strongly suggest that small, monomeric protein enzymes have evolved complex networks of cooperative conformational changes that coordinate the inherently stochastic conformational fluctuations of multiple structural elements in a manner that is optimal for catalysis (2). Within the context of energy landscape theory (3), such enzymes can be thought of as having evolved dynamic energy landscapes that bias enzyme conformational sampling in such a manner to maximize the efficiency of catalysis (2). Related proposals suggest that binding of allosteric effectors to enzymes remodels such energy landscapes, altering enzyme conformational sampling as part of the mechanisms through which these effectors regulate enzymatic activity (4).Unfortunately, the conformational dynamics of large, macromolecular complexes remain much more challenging to characterize than those of small, monomeric proteins (5), making it very difficult to elucidate the role that cooperative conformational changes play in the function and regulation of biomolecular machines such as the ribosome (6). Following each round of aminoacyl–tRNA incorporation and peptide bond formation by the translating ribosome, the resulting ribosomal pretranslocation (PRE) complex must rapidly translocate the newly deacylated tRNA from the ribosomal peptidyl–tRNA binding (P) site to the ribosomal deacylated (or exit) tRNA binding (E) site and the newly formed peptidyl–tRNA from the ribosomal aminoacyl–tRNA binding (A) site to the P site. Concurrent with translocation of the tRNAs, the PRE complex advances along the mRNA by precisely one codon and ultimately ejects the E-site tRNA, producing a ribosomal posttranslocation (POST) complex that is ready for the next round of the elongation cycle.During the first step of translocation, the A- and P-site tRNAs are reconfigured from their classical P/P (denoting the small ribosomal subunit P site/large ribosomal subunit P site) and A/A configurations into their hybrid P/E and A/P configurations. Numerous studies suggest that formation of the hybrid tRNA configuration is accompanied by large-scale conformational changes of the ribosome itself (6–9). Indeed, relative to X-ray crystallographic and cryogenic electron microscopy (cryo-EM) structures of ribosomal complexes carrying classically configured tRNAs, structures of ribosomal complexes carrying hybrid-configured tRNAs exhibit a ratchet-like relative rotation of the ribosomal subunits as well as a closure of the L1 stalk element of the large subunit [defined as 23S ribosomal RNA (rRNA) helices 76, 77, and 78 (H76–78) and ribosomal (r)-protein L1] that allows the L1 stalk to establish a physical interaction with the P/E-configured tRNA (10, 11). Notably, single-molecule fluorescence resonance energy transfer (smFRET) studies have shown that in the absence of elongation factor G (EF-G), the guanosine triphosphatase translation factor that promotes further steps along the translocation pathway, PRE complexes and PRE complex analogs lacking an A-site tRNA (PRE^–A complexes) undergo thermally activated, stochastic, and reversible fluctuations between classical and hybrid tRNA configurations (classical⇆hybrid) (12), nonrotated and rotated subunit orientations (NR⇆R) (13), and open and closed L1 stalk conformations (L1_o⇆L1_c) (14, 15). Unfortunately, however, whether and how these stochastic tRNA, intersubunit, and L1 stalk dynamics are coordinated within PRE/PRE^–A complexes to facilitate translocation and/or allosteric regulation of translocation remains unknown, severely limiting our understanding of the fundamental physical processes that drive and control the rapid and precise translocation of the tRNAs through the ribosome during protein synthesis.To determine whether tRNA, intersubunit, and L1 stalk movements are coordinated within PRE/PRE^–A complexes and to characterize the structural basis for such cooperative conformational changes, here we have used smFRET to characterize the L1 stalk and tRNA dynamics of Escherichia coli PRE^–A complexes assembled using ribosomes that have been strategically mutagenized or treated with a ribosome-binding inhibitor to predictably perturb intersubunit rotation. Together with the results of complementary translocation studies, our findings directly demonstrate that the ribosome uses cooperative conformational changes to maximize and regulate the efficiency of translocation and E-site tRNA ejection during translation. Using structural and phylogenetic analyses of ribosomes to rationalize our results leads us to propose a structure-based model for the observed cooperativity. Collectively, the work presented here strongly suggests that coordination of spatially remote conformational changes is a fundamental aspect of the mechanism and regulation of translocation, other steps of the translation elongation cycle, other stages of protein synthesis, and other biomolecular machines.     

The contributions of individual countries and regions to the global radiative forcing

Knowing the historical relative contribution of greenhouse gases (GHGs) and short-lived climate forcers (SLCFs) to global radiative forcing (RF) at the regional level can help understand how future GHGs emission reductions and associated or independent reductions in SLCFs will affect the ultimate purpose of the Paris Agreement. In this study, we use a compact Earth system model to quantify the global RF and attribute global RF to individual countries and regions. As our evaluation, the United States, the first 15 European Union members, and China are the top three contributors, accounting for 21.9 ± 3.1%, 13.7 ± 1.6%, and 8.6 ± 7.0% of global RF in 2014, respectively. We also find a contrast between developed countries where GHGs dominate the RF and developing countries where SLCFs including aerosols and ozone are more dominant. In developing countries, negative RF caused by aerosols largely masks the positive RF from GHGs. As developing countries take measures to improve the air quality, their negative contributions from aerosols will likely be reduced in the future, which will in turn enhance global warming. This underlines the importance of reducing GHG emissions in parallel to avoid any detrimental consequences from air quality policies.

Anthropogenic activities have been the main drivers of climate change since industrialization, and recent climate change has had substantial impact on both humans and natural systems (1). Increase in anthropogenic greenhouse gas (GHG) emissions is the dominant cause of observed climate warming, and those emissions are driven by the energy demand from economic and population growth since the preindustrial era. In addition to GHGs, anthropogenic activities also change the climate through aerosol emissions, such as black carbon (BC) and organic aerosols (both primary and secondary), as well as aerosol and ozone precursor emissions such as SO₂, NH₃, NO_X, VOC, and CO, all of which are termed short-lived climate forcers (SLCFs). Albedo changes are another climate forcer, mainly induced by land-cover change (LCC). Radiative forcing (RF), a natural or anthropogenic perturbation to the earth’s energy budget, is used to quantify the magnitude by which forcers like GHGs and SLCFs change the climate (2).The Paris Agreement, for the first time, brings all nations into a common cause to undertake ambitious efforts to combat climate change and adapt to its effects. Its goal is to limit global warming to well below 2 °C, preferably to 1.5 °C, compared to preindustrial levels. To achieve this goal, individual signatory countries are requested to submit nationally determined contributions (NDCs) every 5 y. Knowing the historical relative contribution of GHGs and SLCFs to global RF at the regional level can help understand how future GHGs emission reductions and associated or independent reductions in SLCFs will affect the ultimate purpose of the Paris Agreement. Since NDCs are submitted without negotiations and given that stock takes of global emissions and commitments are planned in 2023 and 2028 to assess progress toward the Paris Agreement climate goals, it is even more essential to monitor the contributions of individual countries to climate change.Quantifying the individual country and regional historical contributions to global RF was suggested years ago in the Brazilian Proposal (3) to follow the principle of common but differentiated responsibilities and respective capabilities established by the United Nations Framework Convention on Climate Change (UNFCCC). The Copenhagen Conference of Parties in 2009 marked a shift away from this top-down approach, which meant to attribute mitigation responsibilities relatively to each country’s historical contribution to global climate change, and instead all parties involved in the Paris Agreement in 2016 agreed to take a bottom-up approach yet with a global stock take and a process to improve bottom-up intentions of mitigation.In this study, we quantify individual countries’ and regional contributions to the global RF, following a method close to the one established in our previous estimation for China (3). Compared to this previous work, secondary organic aerosols (SOA), aerosol–cloud interaction, and albedo change induced by BC deposition on snow are included in this study, making it more systematic and comprehensive, albeit at the cost of using a simplified model leading to increased uncertainty. The global RFs are calculated using the reduced-complexity Earth system model OSCAR v3.1, enabled by more complex Earth system models (ESMs) upon which its parameters are calibrated (4–6). The individual countries’ or regional contributions are isolated by using factorial simulations in which a small fraction of each country’s or region’s emissions are removed to quantify their marginal effect on the climate system. This attribution method is used in previous studies (3, 7–9), which is referred to as the “normalized marginal attribution method” (see Methods for details).     

The characteristics of the HLA antigens determined by cellular techniques

The complexity of the D region of the HLA complex is rather great. Three loci--HLA-DP, DQ, DR are very well established in this time. However, the question about the independence of the first known locus of this region--HLA-D--remains elusive--it is still not known if HLA-D antigens are really independent or they rather represent some epitopes of HLA-DR/DQ antigens. The HLA-DP antigens represent the additional series of specificities of the HLA complex recognized by PLT technique. On the other hand HLA-PL antigens, detected by PLT too, correlate as to HLA-D as to HLA-DR antigens and with a great probability they do not form an independent group of HLA antigens.     

Brown adipose tissue: contributions of nature and nurture to the obesity of an obese mutant mouse (ob/ob)

The aim of this investigation was to compare the contributions of the genotype of the brown adipose tissue (BAT) and of its environment to the obesity of the mutant mouse C57 BL/6J ob/ob. Pieces of interscapular BAT from lean or obese mice were transplanted to a site underneath the kidney capsule of recipient lean or obese mice. The grafts were left in place for 6 to 12 weeks and then examined by histological methods by electron microscopy to examine the ultrastructure of the mitochondria and by fluorescence histochemistry to examine the catecholaminergic innervation of the grafts. When lean BAT was grafted into obese mice, or when obese BAT was grafted into lean mice, kept at ambient temperatures, the characteristics of the donor BAT (i.e. lipid droplet size, mitochondrial ultrastructure and catecholaminergic innervation) transformed partially, but not completely, towards those of BAT in the host mouse. However, if lean mice containing obese BAT grafts were cold-acclimated at 4 degrees C or obese mice containing lean BAT grafts were warm-acclimated at 33 degrees C, the characteristics of the donor BAT transformed completely towards those of the BAT in the host mouse. This complete transformation occurred even if the host mice were returned to 23 degrees C after the period of temperature acclimation. Fluorescent histochemical observations indicated that the sympathetic innervation of BAT grafts was only indistinguishable from that of the lean or obese host BAT when the mice received a period of temperature acclimation (cold for lean mice; warm for obese mice). We conclude that BAT grafts from lean mice can assume the typical characteristics of BAT in obese hosts and that BAT grafts from obese mice can assume the typical characteristics of BAT in lean hosts provided that both the sympathetic innervation and the vascularization of the grafts is the same as in the host. Intrinsic properties of BAT in genetically obese mice are therefore unlikely to be of paramount importance in determining the obesity of the ob/ob mouse. Our results support the conclusions of other workers in implicating the low activity of the sympathetic innervation of BAT as being crucially important in causing the reduction of thermogenic activity.     

Electrostatics of nanosystems: application to microtubules and the ribosome

Evaluation of the electrostatic properties of biomolecules has become a standard practice in molecular biophysics. Foremost among the models used to elucidate the electrostatic potential is the Poisson-Boltzmann equation; however, existing methods for solving this equation have limited the scope of accurate electrostatic calculations to relatively small biomolecular systems. Here we present the application of numerical methods to enable the trivially parallel solution of the Poisson-Boltzmann equation for supramolecular structures that are orders of magnitude larger in size. As a demonstration of this methodology, electrostatic potentials have been calculated for large microtubule and ribosome structures. The results point to the likely role of electrostatics in a variety of activities of these structures.     

Structure of the translating Neurospora ribosome arrested by cycloheximide

Ribosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of structural data for actively translating polyribosomes stalled by CHX leaves unanswered the question of which elongation step is inhibited. We elucidated CHX’s mechanism of action based on the cryo-electron microscopy structure of actively translating Neurospora crassa ribosomes bound with CHX at 2.7-Å resolution. The ribosome structure from this filamentous fungus contains clearly resolved ribosomal protein eL28, like higher eukaryotes but unlike budding yeast, which lacks eL28. Despite some differences in overall structures, the ribosomes from Neurospora, yeast, and humans all contain a highly conserved CHX binding site. We also sequenced classic Neurospora CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX resistance in eukaryotes, were in the large subunit proteins uL15 and eL42 that are part of the CHX-binding pocket. In addition to A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX that are associated with the translating N. crassa ribosome, spermidine is present near the CHX binding site close to the E site on the large subunit. The tRNAs in the peptidyl transferase center are in the A/A site and the P/P site. The nascent peptide is attached to the A-site tRNA and not to the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE translocation state and does not interfere with A-site reactivity.

Cycloheximide (CHX) is the most widely used laboratory inhibitor of eukaryotic protein synthesis (1). Alma J. Whiffen, a mycologist working at Upjohn, initially identified it in 1946 as a product of Streptomyces griseus fermentation that she named actidione (diketone produced by an actinomycete); actidione inhibited the growth of fungi but not bacteria (2–4). In 1963, CHX’s function to inhibit eukaryotic protein synthesis was demonstrated using a cell-free translation system (CFTS) from Saccharomyces pastorianus (5). The first induced mutations conferring CHX resistance were isolated and genetically mapped in the model fungus Neurospora crassa (6). Around 2 y later, the direct action of CHX to inhibit translation in a mammalian system was demonstrated using active translation lysates prepared from anucleate rabbit reticulocytes (7). The early literature on CHX and related molecules has been thoroughly and contemporaneously reviewed (8).Structures of the Saccharomyces cerevisiae ribosome (9) and the human ribosome (10), containing CHX tightly bound at the ribosome E site, have given significant insight into its mechanism of action. Based on the position of the CHX binding site, CHX is proposed to interfere with the translocation of P-site transfer RNA (tRNA) to the E site. However, both of these CHX-containing structures were obtained using vacant ribosomes lacking tRNA and therefore did not establish the specific step(s) in the translation cycle at which CHX inhibits elongation. If E-site translocation is blocked as predicted, then this structure is consistent with ribosomes in the pretranslocation (PRE) state with either the nascent peptide on the P-site tRNA and the incoming amino acid on A-site tRNA or in the PRE state with the nascent peptide transferred to A-site tRNA. Both models appear in the literature, with single-molecule biophysical studies supporting the latter (1, 11). Furthermore, the ribosome subunits can have different, relative rotational orientations even while containing A/A and P/P tRNA: A classical PRE state can be discriminated from a rotated PRE* state (12). Thus, there are a variety of states in which CHX could potentially arrest the ribosome even prior to tRNA translocation.Here, we determined the cryo-electron microscopy (cryo-EM) structure of translating N. crassa ribosomes with CHX at 2.7-Å resolution. To accomplish this, we isolated polysomes from actively growing cells to which CHX was added and maintained CHX at a high concentration throughout all subsequent procedures until the vitrification step of cryo-EM sample preparation. We built a model for the N. crassa ribosome that differs from S. cerevisiae ribosomes and more closely resembles higher-eukaryotic ribosomes in that it contains ribosomal protein eL28. Comparisons of structures with CHX bound to ribosomes showed that the CHX-binding position is highly conserved in N. crassa, S. cerevisiae, and human ribosomes. However, unlike the canonical CHX-bound structures of vacant yeast and human ribosomes (9, 10), which contained an Mg²⁺ ion adjacent to the CHX-binding pocket, the N. crassa ribosome contained spermidine (SPD) in place of the Mg²⁺ ion. We sequenced previously identified N. crassa mutations that confer CHX resistance. All amino acid changes in these mutants, including an amino acid change at a previously unidentified position among eukaryotic mutations that confer CHX resistance, map to conserved residues in the CHX-binding pocket. In addition, P/P- and A/A-site tRNAs were present in the N. crassa structure, and the nascent peptide was resolved on the A-site tRNA. Finally, CHX did not appear to interfere with termination as it does with elongation in an N. crassa cell-free translation extract, consistent with CHX interfering with the translocation of tRNA to the E site but not peptidyl transfer events at the A site. The observation that terminating ribosomes are not arrested by CHX could be significant for analyses of the levels of ribosomes mapping to termination codons when CHX is included in ribosome-profiling analyses.This work provides a structural basis for a mechanistic understanding of CHX action. It also provides a high-resolution model for a fungal ribosome that differs from the S. cerevisiae ribosome.