On the Reactivity o f PyridoxaI-5'-phosphate with Yeast tRNAPhe and tRNATyr

Yeast tRNAPhe, Yeast tRNATyr, Pyridoxal-5'-phosphate Yeast tRNAPhe and tRNATyr were reacted with the fluo­ rescent reagent pyridoxal-5'-phosphate and the modified tRNAs were analysed with respect to the number and posi­ tion of modified nucleosides and with respect to aminoacylation. a) Following the intrinsic fluorescence of pyridoxal-5'phosphate, the treatment of tRNATyr with increasing amounts of pyridoxal-5'-phosphate revealed about 50 mol of reagent or a even higher number bound per one mol of tRNATyr. After borohydride reduction (in order to stabilize the linkage) of this modified tRNATyr and purifi­ cation with reverse phase chromatography a modified tRNATyr was obtained carrying about 2 mol of the reagent. b) Both tRNATyr and tRNAPhe treated with pyridoxal5'-phosphate and reduced exhibited almost unchanged aminoacylation as compared to the unmodified tRNAs. c) Pyridoxal-5'-phosphate treated and reduced tRNAPhe and tRNATyr were digested with ribonuclease Tj and the resulting oligonucleotides were separated. However, no flu­ orescent oligonucleotide and no difference to an oligo­ nucleotide pattern obtained from unmodified tRNA were observed. Thus, pyridoxal-5'-phosphate might have been bound to the highly purified yeast tRNAPhe and tRNA1*1' samples either via an unstable linkage or not covalently. This result is controversial with respect to the specific reaction of pyridoxal-5'-phosphate with unfractionated tRNAs from colon carcinoma and tRNAs from E. coli as reported in the literature.