9] Modification of the rare nucleoside x in escherichia coli tRNA with antigenic determining, photolabile, and paramagnetic residues

Abstract

Publisher Summary The chapter discusses the modification of the rare nucleoside X in Escherichia coli tRNAs with antigenic determining, photolabile, and paramagnetic residues. The 3-amino-3-carboxypropyl side chain of X, bearing an aliphatic amino group, is a very good target for covalent attachment of different activated carboxylic acid derivatives resulting in amide bond formation. The specificity of the reaction is due to the strong nucleophilicity of that amino group compared to other residues in the polynucleotide chain. The acylation conditions are simple: the reactions are performed in dimethylsulfoxide (DMSO)/sodium phosphate buffer, pH 8, or DMSO/sodium acetate buffer, pH 7.0, at 40°-50 ° with the appropriate N-hydroxysuccinimide esters. Modified and unmodified tRNA is easily separated. The modified tRNAs are hydrolyzed by T2 RNase, and the digestion mixture is separated by two-dimensional thin-layer chromatography for analysis. The materials of the new spots are isolated, dephosphorylated with alkaline phosphomonoesterase, and compared with authentic synthetic samples of nucleoside derivatives. The modified tRNAs do not accept phenylalanine in the aminoacylation reaction; only partly modified tRNAs can still be partly aminoacylated. Parallel experiments with E. coli tRNA Tyr and tRNA fMet containing no nucleoside X showed no loss of amino acid acceptance after incubation with the N-hydroxysuccinimide ester.