Studies on polynucleotides. CI. Escherichia coli tyrosine and formylmethionine transfer ribonucleic acids: effect of chemical modification of 4-thiouridine to uridine on their biological properties.

A method which uses cyanogen bromide for the quantitative conversion of 4-thiouridine to uridine has been developed. The general nature and the specificity of this reaction has been studied with thiouridine residues present in a hexanucleotide isolated from enzymic digests of Escherichia coli tyrosine transfer ribonucleic acid (tRNA), and also with those present in intact E. coli tyrosine and formylmethionine specific tRNAs. Comparison of enzymic digests of tRNAs before and after treatment with cyanogen bromide showed that the conversion of 4-thiouridine was essentially quantitative. The function of 4-thiouridine in these E. coli tRNAs has been studied by comparing the biological properties of cyanogen bromide-treated tRNAs with those of native tRNAs. For tyrosine tRNA, the rate, extent, and specificity of amino acid acceptance, pyrophosphorolysis or repair of the 3’-terminal-CpCpA by tRNA pyrophosphorylase, codon-directed binding to ribosomes, amino acid incorporation studies in vitro using poly[r(U-A-C)] as messenger RNA, and absorbance versus temperature profiles were compared. Similarly, for the formylmethionine tRNA, the amino acid acceptor activity, the ability for enzymic formylation subsequent to aminoacylation and the ability to initiate protein synthesis in uifro using poly[r(U-G)] as messenger RNA were compared. With the exception of reduced binding of tyrosyltRNATY’ to ribosomes, the results show that 4-thiouridine residues can be converted to uridine without causing any other appreciable change in the properties of these tRNAs.