On the reactivity of pyridoxal-5'-phosphate with yeast tRNAPhe and tRNATyr.

Yeast tRNAPhe and tRNATyr were reacted with the fluorescent reagent pyridoxal-5'-phosphate and the modified tRNAs were analysed with respect to the number and position 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 or 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 purification with reverse phase chromatography a modified tRNATyr was obtained carrying about 2 mol of the reagent. b) Both tRNATyr and tRNAPhe treated with pyridoxal-5'-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 T1 and the resulting oligonucleotides were separated. However, no fluorescent oligonucleotide and no difference to an oligonucleotide pattern obtained from unmodified tRNA were observed. Thus, pyridoxal-5'-phosphate might have been bound to the highly purified yeast tRNAPhe and tRNATyr 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.