Structure of the mammalian ribosome-Sec61 complex to 3.4 Å resolution.

Cotranslational protein translocation is a universally conserved process for secretory and membrane protein biosynthesis. Nascent polypeptides emerging from a translating ribosome are either transported across or inserted into the membrane via the ribosome-bound Sec61 channel. Here, we report structures of a mammalian ribosome-Sec61 complex in both idle and translating states, determined to 3.4 and 3.9 Å resolution. The data sets permit building of a near-complete atomic model of the mammalian ribosome, visualization of A/P and P/E hybrid-state tRNAs, and analysis of a nascent polypeptide in the exit tunnel. Unprecedented chemical detail is observed for both the ribosome-Sec61 interaction and the conformational state of Sec61 upon ribosome binding. Comparison of the maps from idle and translating complexes suggests how conformational changes to the Sec61 channel could facilitate translocation of a secreted polypeptide. The high-resolution structure of the mammalian ribosome-Sec61 complex provides a valuable reference for future functional and structural studies.

Initiation of translation by cricket paralysis virus IRES requires its translocation in the ribosome.

The cricket paralysis virus internal ribosome entry site (CrPV-IRES) is a folded structure in a viral mRNA that allows initiation of translation in the absence of any host initiation factors. By using recent advances in single-particle electron cryomicroscopy, we have solved the structure of CrPV-IRES bound to the ribosome of the yeast Kluyveromyces lactis in both the canonical and rotated states at overall resolutions of 3.7 and 3.8 Å, respectively. In both states, the pseudoknot PKI of the CrPV-IRES mimics a tRNA/mRNA interaction in the decoding center of the A site of the 40S ribosomal subunit. The structure and accompanying factor-binding data show that CrPV-IRES binding mimics a pretranslocation rather than initiation state of the ribosome. Translocation of the IRES by elongation factor 2 (eEF2) is required to bring the first codon of the mRNA into the A site and to allow the start of translation.

Structural changes enable start codon recognition by the eukaryotic translation initiation complex.

During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.

eIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining.

Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.

Selective TnsC recruitment enhances the fidelity of RNA-guided transposition.

Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD1, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs2,3. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins4. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.

Publisher Correction: Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system.

Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements1-3. Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas34,5, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons6,7. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the CRISPR RNA (crRNA). The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.

Quo Vadis CO2 Activation: Catalytic Reduction of CO2 to Methanol Using Aluminum and Gallium/Carbon-based Ambiphiles.

We report on so-called "hidden FLPs" (FLP: frustrated Lewis pair) consisting of a phosphorus ylide featuring a group 13 fragment in the ortho position of a phenyl ring scaffold to form five-membered ring structures. Although the formation of the Lewis acid/base adducts was observed in the solid state, most of the title compounds readily react with carbon dioxide to provide stable insertion products. Strikingly, 0.3-3.0 mol% of the reported aluminum and gallium/carbon-based ambiphiles catalyze the reduction of CO2 to methanol with satisfactory high selectivity and yields using pinacol borane as stoichiometric reduction equivalent. Comprehensive computational studies provided valuable mechanistic insights and shed more light on activity differences.

Metal Influence on Cyaphide-Azide 1,3-Dipolar Cycloaddition Reactions: Aromaticity and Activation Strain.

The factors governing 1,3-dipolar cycloaddition reactions involving C≡P-containing compounds are computationally explored in detail using quantum chemical tools. To this end, the parent process involving tBuN3 and tBuCP is analyzed and compared to the analogous reaction involving organometallic cyaphide complexes (metal=Au, Pt, Ge, Mg), in order to understand the role of the metal fragment in such transformations. It is found that while the metal fragment does not significantly influence the aromaticity of the corresponding concerted transition states or the regioselectivity of the transformation, it may modify the reactivity of the cyaphide complexes (i. e. Ge and Mg cyaphide complexes are comparatively more reactive). The computed reactivity trends and the factors behind the regioselectivity of the cycloaddition reaction are quantitatively analyzed with the help of the activation strain model in combination with the energy decomposition analysis method.

Predisposition in Dynamic Covalent Chemistry: The Role of Non-Covalent Interactions in the Assembly of Tetrahedral Boronate Cages.

Directional bonding strategies guide the design of complex molecular architectures, yet challenges arise due to emergent behavior. Rigid structures face geometric constraints and sensitivity to mismatches, hindering the efficient assembly of molecular organic cages (MOCs). Harnessing intramolecular non-covalent interactions offers a promising solution, broadening geometrical possibilities and enhancing adaptability to boost assembly yields. However, identifying these interactions remains challenging, with their full potential sometimes latent until final assembly. This study explores these challenges by synthesizing boronic acid tripods with varied oxygen positions at the tripodal feet and investigating their role in assembling tetrahedral boronate MOCs. Our results reveal substantial differences in the assembly efficiency among tripods. While the building blocks with oxygen in the benzylic position relative to the central aromatic ring form the MOCs in high yields, those with the oxygen atom directly bound to the central aromatic ring, only yield traces. Through X-ray crystallography and DFT analyses, we elucidate how intramolecular interactions profoundly influence the geometry of the building blocks and cages in a relay-like fashion, highlighting the importance of considering intramolecular interactions in the rational design of (supra)molecular architectures.

Mono(Lewis Base)-Stabilized Gallium Iodide: An Unexplored Class of Promising Ligands.

Quantum-chemical (DFT) calculations on hitherto unknown base(carbene)-stabilized gallium monoiodides (LB→GaI) suggest that these systems feature one lone pair of electrons and a formally vacant p-orbital - both centered at the central gallium atom - and exhibit metallomimetic behavior. The calculated reaction free energies as well as bond dissociation energies suggest that these LB→GaI systems are capable of forming stable donor-acceptor complexes with group 13 trichlorides. Examination of the ligand exchange reactions with iron and nickel complexes indicates their potential use as ligands in transition metal chemistry. In addition, it is found that the title compounds are also able to activate various enthalpically robust bonds. Further, a detailed mechanistic investigation of these small molecule activation processes reveals the non-innocent behavior of the carbene (base) moiety attached to the GaI fragment, thereby indicating the cooperative nature of these bond activation processes. The energy decomposition analysis (EDA) and activation strain model (ASM) of reactivity were also employed to quantitatively understand and rationalize the different activation processes.

A Deeper Insight into the Supramolecular Activation of Oxidative Addition Reactions Involving Pincer-Rhodium(I) Complexes.

The factors governing the acceleration of the oxidative addition of methyl iodide to pincer rhodium(I)-complexes induced by coronene have been computationally explored in detail using quantum chemical methods. Both the parent reaction and the coronene-mediated process proceed via a stepwise SN2-type mechanism. It is found that the acceleration of the process derives from the formation of an initial supramolecular complex, mainly stabilized by electrostatic and π-π interactions, which significantly increases the electron richness of the complex. The impact of this effect on the reaction barrier has been quantitatively analyzed by applying the activation strain model in combination with the energy decomposition analysis method. In addition, the influence of other polycyclic aromatic hydrocarbons on the oxidative reaction has been also considered.

An Unexpected Lewis Acid-Catalyzed Cascade during the Synthesis of the DEF-Benzoxocin Ring System of Nogalamycin and Menogaril: Mechanistic Elucidation by Intermediate Trapping Experiments and Density Functional Theory Studies.

An unexpected Lewis acid-catalyzed carbohydrate rearrangement of a 1,5-bis-glycopyranoside to the product of a formal intramolecular C-aryl glycosylation reaction is reported. Mechanistic studies based mainly on intermediate trapping experiments and density functional theory (DFT) calculations reveal a cascade process involving three transient (a)cyclic oxocarbenium cations, the breaking of three single C(sp3)-O bonds, and the formation of three single bonds (i.e., exo-, endo-, and C-glycosidic bonds), leading to the 2,6-epoxybenzoxocine skeleton of bioactive natural glycoconjugates related to serjanione A and mimocaesalpin E. DFT calculations established that the generation of the pyran moiety embedded in the bridged benzoxocin ring system is likely to proceed through an unusual ring-closure of an ortho-quinone methide intermediate in which the attacking nucleophile is a carbonyl oxygen.

A Mixed-Valence Ti(II)/Ti(III) Inverted Sandwich Compound as a Regioselective Catalyst for the Uncommon 1,3,5-Alkyne Cyclotrimerization.

The synthesis, structure, and catalytic activity of a Ti(II)/Ti(III) inverted sandwich compound are presented in this study. Synthesis of the arene-bridged dititanium compound begins with the preparation of the titanium(IV) precursor [TiCl2(MesPDA)(thf)2] (MesPDA = N,N'-bis(2,4,6-trimethylphenyl)-o-phenylenediamide) (2). The reduction of 2 with sodium metal results in species [{Ti(MesPDA)(thf)}2(µ-Cl)3{Na}] (3) in oxidation state III. To achieve the lower oxidation state II, 2 undergoes reduction through alkylation with lithium cyclopentyl. This alkylation approach triggers a cascade of reactions, including ß-hydride abstraction/elimination, hydrogen evolution, and chemical reduction, to generate the Ti(II)/Ti(III) compound [Li(thf)4][(TiMesPDA)2(µ-η6: η6-C6H6)] (4). X-ray and EPR characterization confirms the mixed-valence states of the titanium species. Compound 4 catalyzes a mild, efficient, and regiospecific cyclotrimerization of alkynes to form 1,3,5-substituted arenes. Kinetic data support a mechanism involving a binuclear titanium arene compound, similar to compound 4, as the resting state. The active catalyst promotes the oxidative coupling of two alkynes in the rate-limiting step, followed by a rapid [4 + 2] cycloaddition to form the arene product. Computational analysis of the resting state for the cycloaddition of trimethylsilylacetylene indicates a thermodynamic preference for stabilizing the 1,3,5-arene within the space between the two [TiMesPDA] fragments, consistent with the observed regioselectivity.

Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1-40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.

Visible-Light Activation of Diorganyl Bis(pyridylimino) Isoindolide Aluminum(III) Complexes and Their Organometallic Radical Reactivity.

We report on the synthesis and characterization of a series of (mostly) air-stable diorganyl bis(pyridylimino) isoindolide (BPI) aluminum complexes and their chemistry upon visible-light excitation. The redox non-innocent BPI pincer ligand allows for efficient charge transfer homolytic processes of the title compounds. This makes them a universal platform for the generation of carbon-centered radicals. The photo-induced homolytic cleavage of the Al-C bonds was investigated by means of stationary and transient UV/Vis spectroscopy, spin trapping experiments, as well as EPR and NMR spectroscopy. The experimental findings were supported by quantum chemical calculations. Reactivity studies enabled the utilization of the aluminum complexes as reactants in tin-free Giese-type reactions and carbonyl alkylations under ambient conditions, which both indicated radical-polar crossover behavior. A deeper understanding of the physical fundamentals and photochemical process was provided, furnishing in turn a new strategy to control the reactivity of bench-stable aluminum organometallics.

Isonitriles as Alkyl Radical Precursors in Visible Light Mediated Hydro- and Deuterodeamination Reactions.

Herein, we report the use of isonitriles as alkyl radical precursors in light-mediated hydro- and deuterodeamination reactions. The reaction is scalable, shows broad functional group compatibility and potential to be used in late-stage functionalization. Importantly, the method is general for Cα -primary, Cα -secondary and Cα -tertiary alkyl isonitriles. For most examples, high yields were obtained through direct visible-light irradiation of the isonitrile in the presence of a silyl radical precursor. Interestingly, in the presence of an organic photocatalyst (4CzIPN) a dramatic acceleration was observed. In-depth mechanistic studies using UV/Vis absorption, steady-state and time-resolved photoluminescence, and transient absorption spectroscopy suggest that the excited state of 4CzIPN can engage in a single-electron transfer with the isonitrile.

The Israeli acute paralysis virus IRES captures host ribosomes by mimicking a ribosomal state with hybrid tRNAs.

Colony collapse disorder (CCD) is a multi-faceted syndrome decimating bee populations worldwide, and a group of viruses of the widely distributed Dicistroviridae family have been identified as a causing agent of CCD. This family of viruses employs non-coding RNA sequences, called internal ribosomal entry sites (IRESs), to precisely exploit the host machinery for viral protein production. Using single-particle cryo-electron microscopy (cryo-EM), we have characterized how the IRES of Israeli acute paralysis virus (IAPV) intergenic region captures and redirects translating ribosomes toward viral RNA messages. We reconstituted two in vitro reactions targeting a pre-translocation and a post-translocation state of the IAPV-IRES in the ribosome, allowing us to identify six structures using image processing classification methods. From these, we reconstructed the trajectory of IAPV-IRES from the early small subunit recruitment to the final post-translocated state in the ribosome. An early commitment of IRES/ribosome complexes for global pre-translocation mimicry explains the high efficiency observed for this IRES. Efforts directed toward fighting CCD by targeting the IAPV-IRES using RNA-interference technology are underway, and the structural framework presented here may assist in further refining these approaches.

Long-term outcomes of the global tuberculosis and COVID-19 co-infection cohort.

BACKGROUND: Longitudinal cohort data of patients with tuberculosis (TB) and coronavirus disease 2019 (COVID-19) are lacking. In our global study, we describe long-term outcomes of patients affected by TB and COVID-19. METHODS: We collected data from 174 centres in 31 countries on all patients affected by COVID-19 and TB between 1 March 2020 and 30 September 2022. Patients were followed-up until cure, death or end of cohort time. All patients had TB and COVID-19; for analysis purposes, deaths were attributed to TB, COVID-19 or both. Survival analysis was performed using Cox proportional risk-regression models, and the log-rank test was used to compare survival and mortality attributed to TB, COVID-19 or both. RESULTS: Overall, 788 patients with COVID-19 and TB (active or sequelae) were recruited from 31 countries, and 10.8% (n=85) died during the observation period. Survival was significantly lower among patients whose death was attributed to TB and COVID-19 versus those dying because of either TB or COVID-19 alone (p<0.001). Significant adjusted risk factors for TB mortality were higher age (hazard ratio (HR) 1.05, 95% CI 1.03-1.07), HIV infection (HR 2.29, 95% CI 1.02-5.16) and invasive ventilation (HR 4.28, 95% CI 2.34-7.83). For COVID-19 mortality, the adjusted risks were higher age (HR 1.03, 95% CI 1.02-1.04), male sex (HR 2.21, 95% CI 1.24-3.91), oxygen requirement (HR 7.93, 95% CI 3.44-18.26) and invasive ventilation (HR 2.19, 95% CI 1.36-3.53). CONCLUSIONS: In our global cohort, death was the outcome in >10% of patients with TB and COVID-19. A range of demographic and clinical predictors are associated with adverse outcomes.

Understanding the Reactivity of Diazaborinines towards the Activation of σ-Bonds.

The poorly understood factors governing the small molecule activation reactions mediated by diazaborinines have been computationally explored in detail using quantum chemical tools. To this end, the activation of E-H σ-bonds (E = H, C, Si, N, P, O, S) has been investigated. These reactions, which proceed in a concerted manner, are exergonic and, in general, associated with relatively low activation barriers. In addition, the barrier becomes lower for the E-H bonds involving the heavier element in the same group (ΔG≠ : C>Si; N>P; O>S). This reactivity trend together with the mode of action of the diazaborinine system are quantitatively analyzed by means of the activation strain model of reactivity in combination with the energy decomposition analysis method.