Suppressor [32P]phosphoseryl-tRNA, prepared using bovine seryl-tRNA synthetase and ATP:seryl-tRNA phosphotransferase, was mixed with rabbit reticulocyte lysates containing endogenous hemoglobin mRNA having the termination codon UGA (opal). The chromatographic pattern of the lysate on Sephacryl S-200 showed that the radioactivity of [32P]phosphate in the hot trichloroacetic acid-precipitate (phosphoprotein) was eluted at the position between mature hemoglobin and globin subunits. The phosphoprotein, obtained by chromatography on S-200, moved to the position corresponding to that of globin readthrough protein on SDS-PAGE. The analyses of the hydrolyzate of the phosphoprotein showed the presence of phosphoserine in the protein. These results suggest that animal opal suppressor tRNA functions in vitro to transfer phosphoserine to the position of the termination codon UGA (opal) on mRNA.
Amino acid residues contained in the recognition sites of seryl-transfer ribonucleic acid (tRNA) synthetase (SerRS) were studied by chemical modification.Ser residues were modified with phenylmethanesulfonyl fluoride, and appeared to be unnecessary for the recognition. However, the modification of Arg residues with phenylglyoxal, His residues with diethylpyrocarbonate and sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, iodoacetic acid or iodoacetamide showed that these residues were essential for the tRNA recognition by SerRS. Protection experiments with substrates from inactivation of Ser-tRNA formation by modification suggested that Arg residues interact with the y-phosphate of adenosine triphosphate and tRNA .Modification of sulfhydryl groups showed that the groups interact with the hydroxyl groups of ribose of the CCA-end on tRNA. Furthermore, in order to understand the recognition mechanism between SerRS and tRNAser, some kinetic parameters such as the Km and V. values of yeast tRNAs" and E. coli tRNAser for bovine SerRS were compared with the values of bovine tRNAser .
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