Hypusination is a unique post-translational modification of the eukaryotic translation factor 5A (eIF5A) that is essential for overcoming ribosome stalling at polyproline sequence stretches. The ...initial step of hypusination, the formation of deoxyhypusine, is catalyzed by deoxyhypusine synthase (DHS), however, the molecular details of the DHS-mediated reaction remained elusive. Recently, patient-derived variants of DHS and eIF5A have been linked to rare neurodevelopmental disorders. Here, we present the cryo-EM structure of the human eIF5A-DHS complex at 2.8 Å resolution and a crystal structure of DHS trapped in the key reaction transition state. Furthermore, we show that disease-associated DHS variants influence the complex formation and hypusination efficiency. Hence, our work dissects the molecular details of the deoxyhypusine synthesis reaction and reveals how clinically-relevant mutations affect this crucial cellular process.
tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cm
U
) in ...eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood. In this study, we present a series of cryo-EM structures of human ELP123 in complex with tRNA and cofactors at four different stages of the reaction. The structures at resolutions of up to 2.9 Å together with complementary functional analyses reveal the molecular mechanism of the modification reaction. Our results show that tRNA binding exposes a universally conserved uridine at position 33 (U
), which triggers acetyl-CoA hydrolysis. We identify a series of conserved residues that are crucial for the radical-based acetylation of U
and profile the molecular effects of patient-derived mutations. Together, we provide the high-resolution view of human Elongator and reveal its detailed mechanism of action.
Tn7 is a bacterial transposon with relatives containing element-encoded CRISPR-Cas systems mediating RNA-guided transposon insertion. Here, we present the 2.7 Å cryoelectron microscopy structure of ...prototypic Tn7 transposase TnsB interacting with the transposon end DNA. When TnsB interacts across repeating binding sites, it adopts a beads-on-a-string architecture, where the DNA-binding and catalytic domains are arranged in a tiled and intertwined fashion. The DNA-binding domains form few base-specific contacts leading to a binding preference that requires multiple weakly conserved sites at the appropriate spacing to achieve DNA sequence specificity. TnsB binding imparts differences in the global structure of the protein-bound DNA ends dictated by the spacing or overlap of binding sites explaining functional differences in the left and right ends of the element. We propose a model of the strand-transfer complex in which the terminal TnsB molecule is rearranged so that its catalytic domain is in a position conducive to transposition.
Abstract
Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional ...addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are crucial for maintaining proteome integrity. In eukaryotes, wobble uridines are modified by Elongator, a large and highly conserved macromolecular complex. Elongator consists of two subcomplexes, namely Elp123 containing the enzymatically active Elp3 subunit and the associated Elp456 hetero-hexamer. The structure of the fully assembled complex and the function of the Elp456 subcomplex have remained elusive. Here, we show the cryo-electron microscopy structure of yeast Elongator at an overall resolution of 4.3 Å. We validate the obtained structure by complementary mutational analyses in vitro and in vivo. In addition, we determined various structures of the murine Elongator complex, including the fully assembled mouse Elongator complex at 5.9 Å resolution. Our results confirm the structural conservation of Elongator and its intermediates among eukaryotes. Furthermore, we complement our analyses with the biochemical characterization of the assembled human Elongator. Our results provide the molecular basis for the assembly of Elongator and its tRNA modification activity in eukaryotes.
Lay Summary
The multi-subunit Elongator complex mediates the addition of a carboxymethyl group to wobble uridines in eukaryotic tRNAs. This tRNA modification is crucial to preserve the integrity of cellular proteomes and to protects us against severe neurodegenerative diseases. Elongator is organized in two distinct modules (i) the larger Elp123 subcomplex that binds and modifies the suitable tRNA substrate and (ii) the smaller Elp456 subcomplex that assists the release of the modified tRNA. The presented cryo-EM structures of Elongator show that the assemblies are very dynamic and undergo conformational rearrangements at consecutive steps of the process. Last but not least, the study provides a detailed reaction scheme and shows that the architecture of Elongator is highly conserved from yeast to mammals.
Graphical Abstract
Graphical Abstract
The Elp123 subcomplex of Elongator captures tRNA substrates and modifies them, whereas the Elp456 assists with the release of the modified tRNAs. Yeast and mouse Elongator are structurally almost identical.
•Ni/Al2O3 catalysts and Al2O3 supports were modified with potassium.•Potassium surface state and stability was investigated by the SR-TAD method.•The beneficial role of potassium addition is strongly ...related to its location in the catalyst.•High resistance to coking of the catalyst results from direct interactions of potassium with the surface of Al2O3 support.
The effect of potassium addition to the Ni/Al2O3 steam reforming catalyst has been investigated on several model systems, including K/Al2O3 with various amounts of alkali promoters (1–4wt% of K2O), a model catalyst 90%NiO-10%Al2O3 promoted with potassium and a commercial catalyst. The potassium surface state and stability were investigated by means of the Species Resolved Thermal Alkali Desorption method (SR-TAD). The activity of the catalysts in the steam reforming of methane and their coking-resistance were also evaluated. The results reveal that the beneficial effect of potassium addition is strongly related to its location in the catalysts. The catalyst surface should be promoted with potassium in order to obtain high coking-resistant catalysts. Moreover, the catalyst preparation procedure should ensure a direct interaction of potassium with the Al2O3 support surface. Due to the low stability of potassium on θ-Al2O3 this phase is undesirable during the preparation of a stable steam reforming catalyst.
Abstract Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural ...proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14’s exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.
Abstract
Betacoronaviruses are a genus within the Coronaviridae family of RNA viruses. They are capable of infecting vertebrates and causing epidemics as well as global pandemics in humans. ...Mitigating the threat posed by Betacoronaviruses requires an understanding of their molecular diversity. The development of novel antivirals hinges on understanding the key regulatory elements within the viral RNA genomes, in particular the 5′-proximal region, which is pivotal for viral protein synthesis. Using a combination of cryo-electron microscopy, atomic force microscopy, chemical probing, and computational modeling, we determined the structures of 5′-proximal regions in RNA genomes of Betacoronaviruses from four subgenera: OC43-CoV, SARS-CoV-2, MERS-CoV, and Rousettus bat-CoV. We obtained cryo-electron microscopy maps and determined atomic-resolution models for the stem-loop-5 (SL5) region at the translation start site and found that despite low sequence similarity and variable length of the helical elements it exhibits a remarkable structural conservation. Atomic force microscopy imaging revealed a common domain organization and a dynamic arrangement of structural elements connected with flexible linkers across all four Betacoronavirus subgenera. Together, these results reveal common features of a critical regulatory region shared between different Betacoronavirus RNA genomes, which may allow targeting of these RNAs by broad-spectrum antiviral therapeutics.
Graphical Abstract
Graphical Abstract
Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome b
6
f (Cyt b
6
f) is known as one of the key steps of photosynthesis, ...but the catalytic mechanism in the plastoquinone oxidation site (Q
p
) remains elusive. Here, we describe two high-resolution cryo-EM structures of the spinach Cyt b
6
f homodimer with endogenous plastoquinones and in complex with plastocyanin. Three plastoquinones are visible and line up one after another head to tail near Q
p
in both monomers, indicating the existence of a channel in each monomer. Therefore, quinones appear to flow through Cyt b
6
f in one direction, transiently exposing the redox-active ring of quinone during catalysis. Our work proposes an unprecedented one-way traffic model that explains efficient quinol oxidation during photosynthesis and respiration.
Structures of cytochrome b
6
f with and without plastocyanin imply a one-way traffic of quinones for efficient photosynthesis.
The enhancement of the technical properties of a Clay/Polymer Nanocomposite (CPN) is related to the homogeneity and dispersion of the filler within the polymer matrix. In this work, samples of pure ...polyamide 6,6 (PA66) and reinforced PA66 with 1, 3, 5, 7 and 9wt.% of sepiolite have been studied. The samples have been qualitatively analyzed with different microscopy techniques and with X-Ray diffraction and scattering techniques. The images obtained by confocal microscopy show that the sepiolite is homogeneously distributed in the PA66 matrix. The micrographs taken by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) show that sepiolite fibres are oriented and equidistantly distributed even in the samples with high percentages of sepiolite. TEM images reveal the absence of clusters of sepiolite and good dispersion of the reinforcement within the matrix. The quantification of the dispersion, calculated from the results of Small Angle X-Ray Scattering (SAXS), indicates that the polymer chains are expanded due to the arrangement of sepiolite within the PA66 matrix and that the fibres are properly dispersed in the polymer.
•Polymer chains are expanded due to the arrangement of sepiolite in injection molded sep/PA66 CPN.•Sepiolite fibers are properly dispersed in the polymer matrix.•SAXS analysis and electron density difference are suitable tools to quantify the dispersion of the filler in CPN.
Copper oxide nanoparticles were dispersed onto the surface of ceria nanorods, while a post‐synthesis modification was applied to the reference sample using ammonia solutions. The modified catalysts ...were characterized with a variety of analytical techniques, providing useful information on the effect of the Cu:NH3 ratio, employed in the modification step, on the physicochemical properties. Re‐dispersion of the active copper species under extremely basic conditions promoted the reducibility and the oxygen mobility of the catalyst, thus resulting in a more active and selective catalyst for preferential CO oxidation.
Selective CO oxidation: CuO‐CeO2 catalysts were modified via a post‐synthesis treatment using ammonia solutions. The catalysts were characterized with a variety of analytical techniques, providing useful information on the effect of the Cu:NH3 ratio on the physicochemical properties. Re‐dispersion of the active copper species under extremely basic conditions promoted the reducibility and the oxygen mobility of the catalyst, thus resulting in a more efficient catalyst for selective CO oxidation.
