Modifications of nucleosides and nucleotides: VI. The reaction of Girard-P reagent with transfer RNA

Abstract

1. Girard-P reagent (acetohydrazide pyridinium chloride) specifically converted the cytidine residues of yeast transfer RNA (tRNA) into 4-deamino-4-acetohydrazidopyridinium cytidine residues without altering any other bases. 2. The tRNA modified with Girard-P reagent was completely hydrolysed by alkali or snake-venom phosphodiesterase (orthophosphoric diester phosphohydrolase, EC 3.1.4.1). A quantitative estimation of the modified cytidine residues in the tRNA was achieved by hydrolysis with the phosphodiesterase and subsequent separation of the nucleotides by ion-exchange column chromatography and estimation of the base composition. 3. The results of column chromatography and sedimentation analysis of the tRNA modified with Girard-P reagent indicated that no splitting of the internucleotide bonds of the tRNA occurred during the reaction. 4. The behavior of the modified tRNA in ion-exchange column chromatography indicated that the tRNA had less negative net charge than the native tRNA after the modification reaction. 5. The temperature-absorbance relationship plotted for the modified tRNA indicated that the secondary structure in the tRNA remarkably decreased after the modification. 6. The effect of the Girard-P modification on the aminoacylation of tRNA for tyrosine, alanine and valine was investigated.