Recycling of Eukaryotic Posttermination Ribosomal Complexes

SummaryAfter translational termination, mRNA and P site deacylated tRNA remain associated with ribosomes in posttermination complexes (post-TCs), which must therefore be recycled by releasing mRNA and deacylated tRNA and by dissociating ribosomes into subunits. Recycling of bacterial post-TCs requires elongation factor EF-G and a ribosome recycling factor RRF. Eukaryotes do not encode a RRF homolog, and their mechanism of ribosomal recycling is unknown. We investigated eukaryotic recycling using post-TCs assembled on a model mRNA encoding a tetrapeptide followed by a UAA stop codon and report that initiation factors eIF3, eIF1, eIF1A, and eIF3j, a loosely associated subunit of eIF3, can promote recycling of eukaryotic post-TCs. eIF3 is the principal factor that promotes splitting of posttermination ribosomes into 60S subunits and tRNA- and mRNA-bound 40S subunits. Its activity is enhanced by eIFs 3j, 1, and 1A. eIF1 also mediates release of P site tRNA, whereas eIF3j ensures subsequent dissociation of mRNA

In Vitro Reconstitution of Eukaryotic Translation Reveals Cooperativity between Release Factors eRF1 and eRF3

SummaryEukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. Whereas eRF1 recognizes all three termination codons and induces hydrolysis of peptidyl tRNA, eRF3's function remains obscure. Here, we reconstituted all steps of eukaryotic translation in vitro using purified ribosomal subunits; initiation, elongation, and termination factors; and aminoacyl tRNAs. This allowed us to investigate termination using pretermination complexes assembled on mRNA encoding a tetrapeptide and to propose a model for translation termination that accounts for the cooperative action of eRF1 and eRF3 in ensuring fast release of nascent polypeptide. In this model, binding of eRF1, eRF3, and GTP to pretermination complexes first induces a structural rearrangement that is manifested as a 2 nucleotide forward shift of the toeprint attributed to pretermination complexes that leads to GTP hydrolysis followed by rapid hydrolysis of peptidyl tRNA. Cooperativity between eRF1 and eRF3 required the eRF3 binding C-terminal domain of eRF1

Dual tRNA mimicry in the Cricket Paralysis Virus IRES uncovers an unexpected similarity with the Hepatitis C Virus IRES

Co-opting the cellular machinery for protein production is a compulsory requirement for viruses. The Cricket Paralysis Virus employs an Internal Ribosomal Entry Site (CrPV-IRES) to express its structural genes in the late stage of infection. Ribosome hijacking is achieved by a sophisticated use of molecular mimicry to tRNA and mRNA, employed to manipulate intrinsically dynamic components of the ribosome. Binding and translocation through the ribosome is required for this IRES to initiate translation. We report two structures, solved by single particle electron cryo-microscopy (cryoEM), of a double translocated CrPV-IRES with aminoacyl-tRNA in the peptidyl site (P site) of the ribosome. CrPV-IRES adopts a previously unseen conformation, mimicking the acceptor stem of a canonical E site tRNA. The structures suggest a mechanism for the positioning of the first aminoacyl-tRNA shared with the distantly related Hepatitis C Virus IRES

AUG_hairpin: prediction of a downstream secondary structure influencing the recognition of a translation start site

<p>Abstract</p> <p>Background</p> <p>The translation start site plays an important role in the control of translation efficiency of eukaryotic mRNAs. The recognition of the start AUG codon by eukaryotic ribosomes is considered to depend on its nucleotide context. However, the fraction of eukaryotic mRNAs with the start codon in a suboptimal context is relatively large. It may be expected that mRNA should possess some features providing efficient translation, including the proper recognition of a translation start site. It has been experimentally shown that a downstream hairpin located in certain positions with respect to start codon can compensate in part for the suboptimal AUG context and also increases translation from non-AUG initiation codons. Prediction of such a compensatory hairpin may be useful in the evaluation of eukaryotic mRNA translation properties.</p> <p>Results</p> <p>We evaluated interdependency between the start codon context and mRNA secondary structure at the CDS beginning: it was found that a suboptimal start codon context significantly correlated with higher base pairing probabilities at positions 13 – 17 of CDS of human and mouse mRNAs. It is likely that the downstream hairpins are used to enhance translation of some mammalian mRNAs <it>in vivo</it>. Thus, we have developed a tool, <it>AUG_hairpin</it>, to predict local stem-loop structures located within the defined region at the beginning of mRNA coding part. The implemented algorithm is based on the available published experimental data on the CDS-located stem-loop structures influencing the recognition of upstream start codons.</p> <p>Conclusion</p> <p>An occurrence of a potential secondary structure downstream of start AUG codon in a suboptimal context (or downstream of a potential non-AUG start codon) may provide researchers with a testable assumption on the presence of additional regulatory signal influencing mRNA translation initiation rate and the start codon choice. <it>AUG_hairpin</it>, which has a convenient Web-interface with adjustable parameters, will make such an evaluation easy and efficient.</p

Couette flow of pentane in clay nanopores: Molecular dynamics simulation

International audienceShear flow of n-pentane in slit nanopores between clay surfaces is investigated by molecular dynamics simulations. Pyrophyllite and hydrated Na-montmorillonite are considered as representative examples of hydrophobic and hydrophilic clay surfaces, respectively. The viscosity-density relations and slip lengths are calculated for both pentane-clay interfaces for different pore widths. The results show that the viscosity-density dependencies for n-pentane are not changed by the confinement in pores with sizes from 3 to 7 nm, compared to the bulk liquid. At the pyrophyllite-pentane interface the slip length is 0.29 nm on average for all studied densities and pore sizes. However, the slip length is negligible at the montmorillonite-pentane interface, likely due to the microscopic roughness of the interface between pentane and the adsorbed water layer. The orientation analysis shows some preference for pentane molecules ordering parallel to the wall surfaces, which is stronger in pyrophyllite pores compared to the montmorillonite, suggesting an influence of the details of fluid-wall interaction on the liquid structure in nanopore

Interaction of Nitrite Ions with Hydrated Portlandite Surfaces: Atomistic Computer Simulation Study

International audienceThe nitrite admixtures in cement and concrete are used as corrosion inhibitors for steel reinforcement and also as anti-freezing agents. The characterization of the protective properties should account for the decrease in the concentration of free NO2− ions in the pores of cement concretes due to their adsorption. Here we applied the classical molecular dynamics computer simulation approach to quantitatively study the molecular scale mechanisms of nitrite adsorption from NaNO2 aqueous solution on a portlandite surface. We used a new parameterization to model the hydrated NO2− ions in combination with the recently upgraded ClayFF force field (ClayFF-MOH) for the structure of portlandite. The new NO2− parameterization makes it possible to reproduce the properties of hydrated NO2− ions in good agreement with experimental data. In addition, the ClayFF-MOH model improves the description of the portlandite structure by explicitly taking into account the bending of Ca-O-H angles in the crystal and on its surface. The simulations showed that despite the formation of a well-structured water layer on the portlandite (001) crystal surface, NO2− ions can be strongly adsorbed. The nitrite adsorption is primarily due to the formation of hydrogen bonds between the structural hydroxyls on the portlandite surface and both the nitrogen and oxygen atoms of the NO2− ions. Due to that, the ions do not form surface adsorption complexes with a single well-defined structure but can assume various local coordinations. However, in all cases, the adsorbed ions did not show significant surface diffusional mobility. Moreover, we demonstrated that the nitrite ions can be adsorbed both near the previously-adsorbed hydrated Na+ ions as surface ion pairs, but also separately from the cations.</jats:p

Atomistic simulations of ettringite and its aqueous interfaces: Structure and properties revisited with the modified ClayFF force field

International audienceEttringite, (Ca6[Al(OH)6]2[SO4]3·nH2O, n = 24–27), is one of the common phases of cement and plays an important role in cement chemistry as the primary cause of sulphate corrosion in Portland cement. Molecular dynamic computer simulations have already been applied earlier to model the crystal structure of ettringite and its interfaces with aqueous salt solutions. A recently developed version of the widely used ClayFF force field allows now to explicitly take into account the bending of M-O-H angles of (M = Al, Ca), leading to a much better agreement of the simulation results with available experimental data. The structure and dynamics of bulk ettringite crystal and its interfaces with NaCl and Na2SO4 aqueous solutions are quantitatively evaluated here for the new modified version of the force field, ClayFF-MOH, and compared with the results obtained with the earlier version, ClayFF-orig. The crystallographic parameters, elastic properties, the structure and dynamics of intracrystalline hydrogen bonding network and the vibrational spectra of ettringite are calculated by classical molecular dynamics simulations and quantitatively compared with available experimental data using both versions of ClayFF. Atomic density profiles for solution species at the ettringite surface, atomic distributions within the crystal-solution interface, and the interfacial diffusional mobility of the species are also calculated and compared. The results clearly demonstrate the importance of the explicit inclusion of M-O-H angular bending terms for accurate modeling of the mineral systems containing structural and interfacial hydroxide groups. The simulation results also show that the application of the new more accurate ClayFF-MOH version of the force field leads to the formation of a stronger hydrogen bonding network structure in the intercolumnar space of the ettringite crystal and at its surface, resulting in a stronger immobilization of the water molecules involved, as well as the ions. The ionic adsorption at the ettringite surface is also generally stronger than it was predicted by the earlier model

Equation of State, Compressibility, and Vibrational Properties of Brucite over Wide Pressure and Temperature Ranges: Atomistic Computer Simulations with the Modified ClayFF Classical Force Field

International audienceThe behavior of brucite over wide ranges of temperatures and pressures is of great interest for fundamental geochemistry and geophysics. Brucite layers and their octahedral Mg(OH)6 structural units constitute an important structural part of layered dense magnesium hydrous silicates (DMHS), which play a major role in mineral equilibria controlling water balance in the subduction zones of the upper mantle. The ClayFF force field was originally developed for atomistic computer simulations of clays and other layered minerals and their hydrated interfaces. The crystallographic parameters of brucite at 25 °C and 1 bar were used, among several others, to develop the original ClayFF parametrization. Its new recent modification, ClayFF-MOH, can more accurately account for the bending of Mg–O–H angles in the brucite structure, and it was used here to test the applicability of this simple classical model over very wide ranges of temperature and pressure well beyond the range of its original implementation (up to 600 °C and 15 GPa). The pressure and temperature dependencies of brucite crystallographic parameters, the compressibility of the crystal lattice, the coefficients of thermal expansion, and the vibrational spectra were calculated in a series of classical molecular dynamics simulations using the ClayFF-MOH model and compared with a diverse set of available experimental data, including X-ray diffractometry, neutron scattering, IR and Raman spectroscopy. These new results demonstrated that ClayFF-MOH, as simple and approximate as it is, can be quite accurate in predicting many mineral properties at subduction zone conditions, which greatly expands the area of its applicability.</jats:p