Abstract
The 3' half molecule of yeast tRNA(Ala) (nucleotides 36-75) was hybridized with a DNA fragment (5'GGAATCGAACC 3') and the hybrid was then digested with E. coli RNase H (from Boehringer). The enzyme can specifically cleave the 3' half molecule at the 3' side of nucleotide Psi(55), thus a fragment C(36)-Psi(55) was prepared. The 3'-terminal T or TPsi of this fragment was removed by one or two cycles of periodate oxidation and beta-elimination. The products were fragments C(36)-T(54) and C(36)-G(53). Three yeast tRNA(Ala) fragments C(56)-A(76), U(55)-A(76) (with Psi(55) replaced by U), U(54)-A(76) (with T(54)Psi(55) replaced by UU) were synthesized and ligated with three prepared fragments (C(36)-Psi(55), C(36)-T(54) and C(36)-G(53)) respectively by T4 RNA ligase. The products were further ligated with the 5' half molecule (nucleotides 1-35). Using this method, one reconstituted yeast tRNA(Ala) (tRNAr) and two yeast tRNA(ALa) analogs: (i) tRNAa with U(55) instead of Psi(55); (ii) tRNAb with U(54)U(55) instead of T(54)Psi(55) 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 T(54) and Psi(55) play an important role in yeast tRNA(Ala) function.
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