酵母丙氨酸转移核糖核酸3′-半分子(36—76)的合成

本文报告用T_4RNA连接酶将相应于酵母丙氨酸转移核糖核酸(tRNA_y~(Ala))3′-半分子(36—76)的三个寡核昔酸大片段——10(36—45)(Ⅰ),12(46—57)(Ⅱ)和19p(58—76)(Ⅲ)——从3′-端向5′-端延伸逐个连接合成了这个tRNA的3′-半分子(36—76)。首先在供受体配比为1:1.5的情况下,采取三步连续反应,即19p(Ⅲ)的5′-磷酸化,然后与12(Ⅱ)的连接和连接反应产物的5′-磷酸化等反应,一次制备分离的方法,以70％的总得率合成了5′-磷酸化的三十一核苷酸(46—76)(Ⅳ)。然后,以(Ⅳ)作为下一步反应的供体和三倍量的10(Ⅰ)连接,以67％的产率合成了具有四十一核苷酸的tRNA_y~(Ala)的3′-半分子(36—76)(Ⅴ)。将这个合成的3′-半分子,5′-磷酸化以后,与天然的5′-半分子连接,人工半合成了tRNA_y~(Ala)整分子,经生物活力测定,这个人工半合成的tRNA_y~(Ala)具有接受[~3H]-丙氨酸、并能将接受的丙氨酸转移到蛋白质分子中去的生物活力。     

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。     

酵母丙氨酸转移核糖核酸的人工全合成

本文报道采用有机合成与酶促合成相结合的方法,按照R.Holley等测定又经他人修正的一级结构,人工全合成了酵母丙氨酸转移核糖核酸(tRNA_y~(Ala))的工作。我们合成的tRNA_y~(Ala)与天然的tRNA_y~(Ala)具有相同的化学组成(含有全部修饰核苷酸)和结构,并有完整的生物活力,即在大鼠肝氨酰基tRNA合成酶的催化下,能接受丙氨酸,而且在兔网织红细胞裂解液体系中能将所携带的丙氨酸参入到蛋白质中去。在进行全合成以前,曾分别进行了两种人工半合成,即将天然的5′半分子与人工的3′半分子和人工的5′半分子与天然的3′半分子进行连接,都取得有生物活力的整分子tRNA_y~(Ala)。据我们了解,这是世界上第一次用人工方法合成的具有生物功能的核糖核酸。     

SYNTHESIS OF THE 3'-HALF MOLECULE OF YEAST ALANINE tRNA

This paper deals with the synthesis of the 3'-half molecule of yeast alanine transfer RNA (tRNA_y~(Ala)) by ligation with T_4 RNA ligase of three component oligonucleotide fragments corresponding to nucleotides 36-45(Ⅰ), 46-57(Ⅱ) and 58-76(Ⅲ) in succession extending from the 3'-end to the 5'-end. First, in a ratio of acceptor to donor at 1.5 to 1, we adopted a method of three successive reactions, namely, the 5'-phosphorylation of the nonadecamer (Ⅲ), ligation with the dodecamer(Ⅱ) and the 5'-phosphorylation of the ligation product formed; with one isolation step and obtained the 5'-phosphorylated 31mer(46-76) (Ⅳ) in an overall yield of 70%. Then the 31met(Ⅵ) as a donor was ligated with 3 times of decamer(Ⅰ) to form the 41met(36-76) (Ⅴ), the 3'-half molecule of tRNA_y~(Ala)). The yield was 67%. After 5'-phosphorylation, (Ⅴ) was ligated with the natural 5'-half molecule to form the semi-synthetic tRNA_y~(Ala)), which was biologically active, i. e. accepting and transferring (~3H)-alanine into p     

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     

修饰核苷酸的生物功能——Ⅱ.酵母tRNA~(Ala)类似物(A_(34)或G_(34)代替I_(34))的合成及生物活性

酵母tRNA~(Ala)的5′-半分子类似物(A_(34)或G_(34)代替I_(34))与3′-半分子在T_4 RNA连接酶催化下连接成酵母tRNA~(Ala)类似物(A_(34)或G_(34)代替I_(34))的整分子。合成的酵母tRNA~(Ala)类似物,在凝胶电泳上具有与天然酵母tRNA~(Ala)相同长度位置,其纯度为95％左右,连接点、末端分析都是对的。测定了这类似物的生物活力,即在大鼠肝氨酰基tRNA合成酶的催化下,接受丙氨酸的能力与天然重组的酵母tRNA~(Ala)没有明显变化,而在兔网织细胞裂解液体系中能将所携带的丙氨酸参入到蛋白质中去的能力,A_(34)代替I_(34)的只有天然或天然重组酵母tRNA~(Ala)的1/3,而G_(34)代替I_(34)的为90％左右。着重讨论了A或G代替I_(34)后,参入活力变化不同的原因。     

TOTAL SYNTHESIS OF YEAST ALANINE TRANSFER RIBONUCLEIC ACID

By a combination of chemical and enzymatic methods, small oligonucleotides with lengths varying from 2 to 8 nucleotides were synthesized from mononucleotides. The small oligonucleotides were then ligated with T_4 RNA ligase into six laxge ligonucleotides (9 to 19 nucleotides long) which were further ligated to form two half molecules with 35 and 41 nucleotides respectively, Finally, the two synthetic half molecules were annealed and ligated to obtain the whole molecule of yeast alanine tRNA (tRNA_y~(Ala)). Prior to this, two semi-syntheses were performed, i. e. ligation of the synthetic 5'-half molecule with the natural 3'-half molecule and that of the natural 5'-half molecule with the synthetic 3'-half molecule.     

BIOLOGICAL FUNCTION OF MODIFIED NUCLEOTIDES IN tRNA MOLECULES——SYNTHESIS AND BIOLOGICAL ACTIVITY OF THE ANALOGUES OF YEAST ALANYL tRNA WITH I_(34) REPLACED BY A_(34) OR G_(34)

Analogues of yeast alanyl tRNA with I_(34) replaced by A_(34) or G_(34) were synthesized. Synthetic analoguesof yeast alanyl tRNT occupy the same position as the natural yeast alanyl tRNA on polyacrylamide gelelectrophoresis, and their purity is about 95% after electrophoresis on a 10% or 20% polyacrylamide gel.The two terminal and nearest neighbour nucleotides of the analogues are all correct. The accepting acti-vity of the synthetic analogues is similar to that of the reconstituted natural yeast alanyl tRNA. The in-corporation activity of alanine into proteins of the synthetic analogues is about 30% of that of the naturalof reconstituted natural yeast alanyl tRNA when I_(34) is replaced by A, and is 90% when I_(34) is replaced byG.The reason of the variation in biological function of the analogues of yeast alanyl tRNA after I_(34) re-placed by A or G was discussed.