| Biological function of modified nucleotides T_(54) and ψ_(55) of yeast tRNA~(Ala) |
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| 作者姓名： | 蒋华琦 金由辛 王德宝 |
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| 作者单位： | State Key Laboratory of Molecular Biology，Shanghai Institute of Biochemistry，Chinese Academy of Sciences，Shanghai 200031，China，State Key Laboratory of Molecular Biology，Shanghai Institute of Biochemistry，Chinese Academy of Sciences，Shanghai 200031，China，State Key Laboratory of Molecular Biology，Shanghai Institute of Biochemistry，Chinese Academy of Sciences，Shanghai 200031，China |
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| 基金项目： | Project supported by the National Natural Science Foundation of China. |
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| 摘 要： | 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 with E. 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 $-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 activi
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| 关 键 词： | yeast tRNA~(Ala) biological function modified nucleotides T_(54) and ψ_(55) |
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