RESUMO
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.
Assuntos
Dicistroviridae/química , Kluyveromyces/química , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/química , RNA Viral/química , Ribossomos/química , Microscopia Crioeletrônica , Dicistroviridae/genética , Kluyveromyces/metabolismo , Modelos Moleculares , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/ultraestrutura , RNA de Transferência/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , RNA Viral/ultraestrutura , Ribossomos/metabolismo , Ribossomos/ultraestruturaRESUMO
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.
Assuntos
Proteínas de Membrana/química , Biossíntese de Proteínas , Ribossomos/química , Animais , Cristalografia por Raios X , Proteínas de Membrana/metabolismo , Modelos Moleculares , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Canais de Translocação SEC , SuínosRESUMO
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.
Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Kluyveromyces/metabolismo , Iniciação Traducional da Cadeia Peptídica , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Códon de Iniciação , Microscopia Crioeletrônica , Modelos Moleculares , Dados de Sequência Molecular , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Alinhamento de SequênciaRESUMO
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.
Assuntos
Adenosina Trifosfatases , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , RNA Bacteriano , Adenosina Trifosfatases/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Especificidade por Substrato , Transposases/metabolismoRESUMO
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.
Assuntos
Fator de Iniciação 1 em Eucariotos , Fatores de Iniciação em Eucariotos , RNA de Transferência de Metionina , Subunidades Ribossômicas , Microscopia Crioeletrônica , Fator de Iniciação 1 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Humanos , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Subunidades Ribossômicas/química , Subunidades Ribossômicas/metabolismo , Imagem Individual de MoléculaRESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Elementos de DNA Transponíveis , DNA Bacteriano/química , Vibrio cholerae/química , Microscopia Crioeletrônica , DNA Bacteriano/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Multimerização Proteica , Estrutura Quaternária de Proteína , RNA Bacteriano/química , Vibrio cholerae/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
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, 0.3-3.0â 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.
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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.
RESUMO
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.
RESUMO
Quantum-chemical (DFT) calculations on hitherto unknown base(carbene)-stabilized gallium monoiodides (LBâGaI) 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 LBâGaI 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.
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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.
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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.
RESUMO
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.
RESUMO
A bare lead atom is a σ-donor ligand capable of linearly bonding and stabilizing two units of a classical polyhydride complex, with a high-valent metal center. As a proof of concept, we have prepared and characterized the µ2-tetrylide complex (PiPr3)2H4OsâPbâOsH4(PiPr3)2 in the reaction of OsH6(PiPr3)2 with Pb{N(SiMe3)2}2. Although the Pb-Os bonds exhibit electrostatic interaction, the main orbital interactions result from two dative σ bonds from the lead atom to the osmium centers. The latter also provide much weaker π-backdonations.
RESUMO
While metal-ligand cooperativity is well-known, studies on element-ligand cooperativity involving main group species are comparatively much less explored. In this study, we computationally designed a few geometrically constrained borylenes supported by different carbenes. Our density functional theory studies indicate that they possess enhanced nucleophilicity as well as electrophilicity, thus rendering them promising candidates for exhibiting borylene-ligand cooperativity. The cooperation between the boron and adjacent carbene centers facilitates different bond activation processes, including the cycloaddition of acetylene across the boron-carbene bond as well as B-H/Si-H bond activation reactions, which have been analyzed in detail. To the best of our knowledge, the borylenes proposed in this study represent the first examples of theoretically proposed geometrically constrained bis(carbene)-stabilized borylenes capable of cooperative activation of enthalpically strong bonds.
RESUMO
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.
Assuntos
COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Pandemias , Iniciação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Viral/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Proteínas não Estruturais Virais/genéticaRESUMO
The synthesis of housanes derivatives from cyclopropenes is described. Under rhodium(II) catalysis, cyclopropenylvinyl carbinols can regioselectively generate a carbene intermediate which undergo an intramolecular cyclopropanation to form a housane, a skeleton with similar ring strain as the cyclopropene precursor. The procedure shows a remarkable broad scope and efficiency. Moreover, the method served to prepare man-made housane-containing terpene derivatives, which are not accessible by Nature.
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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.
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The synthesis of diradical organic compounds has garnered significant attention due to their thermally accessible spin inversion and optoelectronic properties. Yet, preparing such stable structures with high open-shell behavior remains challenging. Herein, we report the synthesis and properties of four π-extended, fused fluorene derivatives with high diradical character, taking advantage of a molecular design where the closed-shell does not include any Clar sextet, comparatively to a maximum of 5 in the corresponding open-shell state. This led to an unusual open-shell triplet ground state with an outstanding singlet-triplet energy difference (ΔEST) of ca. 19 kcal/mol, one of the highest values reported to date for an all-carbon conjugated scaffold. Incorporation of dithiafulvene units at each end of the molecule (at the five-membered rings) furnishes extended tetrathiafulvalenes (TTFs) undergoing reversible oxidations to the radical cation and diradical dication. The various pro-aromatic structures presented herein show highly localized spin density and a limited conjugation due to the confined π-electrons in the aromatic cycles, as supported by 1H NMR, UV-visible, EPR spectroscopy and DFT calculations.