In order to identify the amino acid residues necessary for the selective recognition of the mRNA cap structure by human eukaryotic initiation factor-4E (eIF-4E), which plays a central role in the first step of mRNA translation, we prepared recombinant wild-type and fourteen mutant forms and compared their cap-binding abilities by affinity chromatography. By the direct expression of a synthetic gene encoding human eIF-4E as the soluble form in Escherichia coli and the application on a 7-methylguanosine-5'-triphosphate-Sepharose 4B cap affinity column, pure recombinant eIF-4E was prepared; the optimum pH for the binding of the mRNA cap was 7.5. Among the amino acid residues conserved among various eIF-4E species, each of 14 functional residues was replaced with a nonpolar amino acid (alanine or leucine). All mutant eIF-4E genes, which were constructed by site-directed niutagenesis, were expressed in the same way as the wild type, and their cap-binding abilities were compared with that of the wild type. Consequently, all eight tryptophan residues, Glu103, and two histidine residues at positions 37 and 200 in human recombinant eIF-4E were suggested to be important for the recognition of the mRNA cap structure through direct interaction and/or indirect contributions. Indirect contributions included the construction of the overall protein structure, especially the cap-binding pocket.Keywords: human eIF-4E ; m'G(5')ppp ; mRNA cap structure ; site-directed mutagenesis ; cap binding.Many eukaryotic mRNAs have a common cap structure [m7G(5')ppp(5')N, where N is any nucleotide] at the 5'-terminal portion and its structure plays important roles in stabilizing the mRNA structure and facilitating mRNA binding to ribosomes during initiation [l-31. To allow for the efficient translation of mRNA, however, an interaction is required between the cap structure and eukaryotic initiation factor (eIF)-4 polypeptides consisting of eIF-4A, eIF-4B, eIF-4E, and eIF-4y 143. eIF-4E, which corresponds to the smallest subunit of eIF-4F, has been shown to bind specifically to the mRNA cap structure and has an important function in the first step of protein synthesis; it appears to play a key role in the regulation of translation via phosphorylation [5 -71. Furthermore, recent studies have indicated that eIF-4E participates in the regulation of translation through the interaction with 4E-BP [8].The amino acid sequences of eIF-4Es from yeast [9], human [lo], mouse [ I l l , rabbit 1121, wheat germ [13], and Drosophila [14] have already been reported. Human eIF-4E is a polypeptide of about 25 kDa that contains eight tryptophan residues and its amino acid residues have been remarkably conserved in number and position during evolution among human, mouse, yeast, rabbit, wheat, and Drosophila, which probably reflects the importance for the recognition of the mRNA cap structure [15]. From fluorescence studies at various pH values, however, histidineCorrespondt.nce to T. Ishida, Department of Physical Chemistry, Osaka University of Pharmaceutical Scie...
The adsorption behavior of 2,5-dimercapto-1,3,4-thiadiazole (DMcT) and 2-mercapto-5-methyl-1,3,4-thiadiazole (McMT) on Au and Cu electrode surfaces was studied using a 5 MHz quartz crystal microbalance (QCM), cyclic voltammetry, Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and phase measurement interferometric microscopy (PMIM). Different behaviors were observed for the adsorption of DMcT and McMT on Au and Cu electrodes. Exposing the Au electrode to a McMT solution resulted in the formation of a stable, self-assembled monolayer on the electrode surface. A sharp peak resulting from the reductive desorption (RD) of McMT was observed for McMT chemisorbed on the Au electrode. It was also found that dimer-DMcT (di-DMcT) should be used in order to construct a stable DMcT layer on an Au electrode. Detailed comparisons of charge consumption and mass change during reductive desorption suggest that chemisorbed di-DMcT is monomeric and desorbs completely from the Au electrode in the RD process. However, on a Cu electrode surface, a stable McMT layer could not be constructed. It was also confirmed from PMIM experiments and Raman spectroscopy that DMcT etched copper electrodes, along with concurrent formation of a dimer form of DMcT (di-DMcT). The apparent reason for the different adsorption behaviors between DMcT and McMT is that DMcT is a stronger proton donor and oxidant.
Interaction between copper and 2,5-dimercapto-1,3,4-thiadiazole (DMcT) plays a crucial role in the performance of the DMcT-based polymer composite cathode with a copper current collector. In order to understand this interaction, we employed voltammetry combined with in situ electrochemical quartz crystal microgravimetry (EQCM) and in situ phase measurement interferometric microscope (PMIM), and investigated electrochemical behavior and surface morphologic changes of a copper substrate in nonaqueous solutions without and with DMcT. Voltammetric results show that DMcT inhibits electrodissolution of copper at potentials negative of 1.0 V vs. SSCE. EQCM results suggest that Cu may dissolve chiefly as Cut, which may be stabilized by DMcT by forming a complex, at more positive potentials than 1.0 V. The PMIM images and corresponding data demonstrate that copper dissolution is a nonuniform process and results in a roughness increase of surface in the absence of DMcT, while the presence of DMcT makes the substrate surface remain relatively smooth. Scanning electron micrographs further support the PMIM observations.) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 128.83.63.180 Downloaded on 2015-06-04 to IP ABSTRACT LiC,, the super dense high-pressure phase o f lithium-intercalated graphite, has been tested i n a two-electrode cell vs. Li with an organic electrolyte. A primary capacity o f 910 rnAh/g per carbon atom was observed during the first deintercalation cycle at constant current, almost three times greater than the ideal 372 mAh/g value for the normal saturatedphase LiC,. LiC, also exhibited the desirable characteristic o f a low and flat working voltage profile, and most o f the Li was removed at -18 mV. The first deintercalation cycle also showed weak anomalies which coincide with previously identified phase transitions between high order Li in-plane superlattices. Repeated cycling yielded a reversible capacity close t o 372 mAh/g, with Li removed at -100 mV. The high initial capacity and near ideal reversible secondary capacity suggests that this material could b e useful i n rechargeable batteries requiring a very large first deintercalation capacity.
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