酵母丙氨酸转移核糖核酸5′-半分子(1—35)的合成

本文报道了应用T_4RNA连接酶将酵母丙氨酸转移核糖核酸(tRNA_y~(Ala))5′-半分子中的三个寡核苷酸片段[—13(Ⅰ);14—22(Ⅱ);23—35(Ⅲ)]连接成5′-半分子的工作。由于寡核苦酸片段的纯度高,多核苷酸激酶和T_4RNA连接酶的质量好,采用连续反应的方法,简化了分离步骤,使产物的得率大大提高,二十二核苷酸的连接率是75％,三十五核苦酸的连接率是90％,以第一步反应原料为基数计算,最终产物的总得率是21％。经连接点和末端核苷酸分析,证明它的结构是正确的。将合成的5′-半分子与天然的3′-半分子在T_4RNA连接酶的催化下连接成人工半合成的完整tRNA_y~(Ala),具有接受[~3H]-丙氨酸和将[~3H]-丙氨酸转移到蛋白质中的生物活力。     

SYNTHESIS OF THE 5'-HALF MOLECULE OF YEAST ALANINE tRNA

In this paper, we report the synthesis of the 5'-half molecule of yeast alanine tRNA (tRNA_y~(Ala)) by ligating three oligonucleotide fragments corresponding to the nucleotide sequences 1-13, 14-22 and 23-35 respectively under the catalysis of T_4 RNA ligase (Fig. 1). Because of the high purity of the oligonuclcotide fragments and the excellent quality of T_4RNA ligase and polynucleotide kinase we prepared, the isolation steps were simplified and the overall yields were much higher. The ligating yield of the docosamer (Ⅳ) was 75%, that of the pentatriacontamer (Ⅴ), 90%, and the isolated yield of the final product was 21% calculated on the basis of the tridecamer (Ⅲ) used in the. first reaction. Under the action of T_4 RNA ligase the synthetic 5'-half molecule was joined with the natural 3'-half molecule forming a semi-synthetic tRNA_y~(Ala), which possessed the biological activities of both accepting (~3H)-alanine and incorpprating it into proteins. The correctness of the structure of the synthetic 5