After translational termination, mRNA and P site deacylated tRNA remain associated with ribosomes in post-termination 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 homologue 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 post-termination ribosomes into 60S subunits and tRNA- and mRNA-bound 40S subunits. Its activity is enhanced by eIF3j, eIF1 and eIF1A. eIF1 also mediates release of P-site tRNA, whereas eIF3j ensures subsequent dissociation of mRNA.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
After termination, eukaryotic 80S ribosomes remain associated with mRNA, P-site deacylated tRNA and release factor eRF1, and must be recycled by dissociating these ligands and separating ribosomes ...into subunits. Although recycling of eukaryotic post-termination complexes (post-TCs) can be mediated by initiation factors eIF3, eIF1 and eIF1A (
Pisarev et al., 2007
), this energy-free mechanism can function only in a narrow range of low Mg
2+
concentrations. Here we report that ABCE1, a conserved and essential member of the ATP-binding cassette (ABC) family of proteins, promotes eukaryotic ribosomal recycling over a wide range of Mg
2+
concentrations. ABCE1 dissociates post-TCs into free 60S subunits and mRNA- and tRNA-bound 40S subunits. It can hydrolyze ATP, GTP, UTP and CTP. NTP hydrolysis by ABCE1 is stimulated by post-TCs and is required for its recycling activity. Importantly, ABCE1 dissociates only post-TCs obtained with eRF1/eRF3 (or eRF1 alone), but not post-TCs obtained with puromycin in eRF1's absence.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Eukaryotic protein synthesis begins with assembly of 48S initiation complexes at the initiation codon of mRNA, which requires at least seven initiation factors (eIFs). First, 43S preinitiation ...complexes comprising 40S ribosomal subunits, eIFs 3, 2, 1, and 1A, and tRNA(Met)(i) attach to the 5'-proximal region of mRNA and then scan along the 5' untranslated region (5'UTR) to the initiation codon. Attachment of 43S complexes is mediated by three other eIFs, 4F, 4A, and 4B, which cooperatively unwind the cap-proximal region of mRNA and later also assist 43S complexes during scanning. We now report that these seven eIFs are not sufficient for efficient 48S complex formation on mRNAs with highly structured 5'UTRs, and that this process requires the DExH-box protein DHX29. DHX29 binds 40S subunits and hydrolyzes ATP, GTP, UTP, and CTP. NTP hydrolysis by DHX29 is strongly stimulated by 43S complexes and is required for DHX29's activity in promoting 48S complex formation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Eukaryotic initiation factor (eIF) 1 maintains the fidelity of initiation codon selection and enables mammalian 43S preinitiation complexes to discriminate against AUG codons with a context that ...deviates from the optimum sequence GCC(A/G)CCAUGG, in which the purines at super(-)3 and super(+)4 positions are most important. We hypothesize that eIF1 acts by antagonizing conformational changes that occur in ribosomal complexes upon codon-anticodon base-pairing during 48S initiation complex formation, and that the role of super(-)3 and super(+)4 context nucleotides is to stabilize these changes by interacting with components of this complex. Here we report that U and G at super(+)4 both UV-cross-linked to ribosomal protein (rp) S15 in 48S complexes. However, whereas U cross-linked strongly to C sub(1696) and less well to AA sub(1818-1819) in helix 44 of 18S rRNA, G cross-linked exclusively to AA sub(1818-1819). U at super(-)3 cross-linked to rpS5 and eIF2 alpha , whereas G cross-linked only to eIF2 alpha . Results of UV cross-linking experiments and of assays of 48S complex formation done using alpha -subunit-deficient eIF2 indicate that eIF2 alpha 's interaction with the super(-)3 purine is responsible for recognition of the super(-)3 context position by 43S complexes and suggest that the super(+)4 purine/AA sub(1818-1819) interaction might be responsible for recognizing the super(+)4 position.
Ettringite, (Ca6Al(OH)62SO43·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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The 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 NO
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 NaNO
aqueous solution on a portlandite surface. We used a new parameterization to model the hydrated NO
ions in combination with the recently upgraded ClayFF force field (ClayFF-MOH) for the structure of portlandite. The new NO
parameterization makes it possible to reproduce the properties of hydrated NO
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, NO
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 NO
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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