Biological function of modified nucleotides T54 and Ψ55 of yeast tRNAAla

The 3′ half molecule of yeast tRNAAla (nucleotides 36–75) was hybridized with a DNA fragment (5′GGAATCGAACC 3′) and the hybrid was then digested withE. coli RNase H (from Boehringer). The enzyme can specifically cleave the 3′ half molecule at the 3′ side of nucleotide Ψ55, thus a fragment C36-Ψ55 was prepared. The 3′-terminal T or TΨ of this fragment was removed by one or two cycles of periodate oxidation and β-elimination. The products were fragments C36-T54 and C36-G53. Three yeast tRNAAla fragments C56-A76, U55-A76 (with Ψ55 replaced by U), U54-A76 (with T54Ψ55 replaced by UU) were synthesized and ligated with three prepared fragments (C36-Ψ55, C36-T54 and C36-G53) respectively by T4 RNA ligase. The products were further ligated with the 5′ half molecule (nu-cleotides 1–35). Using this method, one reconstituted yeast tRNAAla (tRNAr) and two yeast tRNAALa analogs: (i) tRNAa with U55 instead of Ψ55; (ii) tRNAb with U54U55 instead of T54Ψ55 were synthesized. The charging and incorporation activities of these three tRNAs were determined. In comparison with the reconstituted tRNA, the charging activity was 75% for tRNAa and 45% for tRNAb and the incorporation activity was 65% for tRNAa and 70% for tRNAb. These results suggest that the modified nucleotides T54 and Ψ55 play an important role in yeast tRNAAla function. Project supported by the National Natural Science Foundation of China.