Koiwai O, Miyazaki M
J Biochem. 1976 Nov;80(5):937-50. doi: 10.1093/oxfordjournals.jbchem.a131381.
The methionine acceptor activity of a crude tRNA from bakers' yeast was resolved into two peaks (I and II) by column chromatography on DEAE-Sephadex A-25 with a 1 M phosphate system. Methionine tRNA from peak II was not formylated by E. coli methionyl-tRNA transformylase [EC 2.1.2.9.] after being charged with methionine, whereas that from peak I was formylatable under the same conditions. A substantial amount of unlabelled methionine tRNA, tRNAMetm, was highly purified from the peak II fraction by successive chromatographic procedures. The purified tRNAMetm was digested with pancreatic ribonuclease A [EC 3.1.4.22] and ribonuclease T1 [EC 3.1.4.8]. The digestion products were isolated into individual components and completely sequenced. The results of sequence analysis of the two RNase digests were in good agreement and indicated that the chain length of this tRNA is 76, including 13 modified nucleotides. These oligonucleotide fragments can be constructed into a unique total sequence, assuming a few conventional features of clover leaf structure for the tRNA was established by analyses of partial digestion products with RNase T1, as reported in the accompanying paper.
用1M磷酸盐系统在DEAE - 葡聚糖A - 25柱上进行层析，可将面包酵母粗tRNA的甲硫氨酸受体活性分离为两个峰（I和II）。来自峰II的甲硫氨酸tRNA在负载甲硫氨酸后，不能被大肠杆菌甲硫氨酰 - tRNA转甲酰酶[EC 2.1.2.9]甲酰化，而来自峰I的甲硫氨酸tRNA在相同条件下可被甲酰化。通过连续的层析步骤，从峰II组分中高度纯化出大量未标记的甲硫氨酸tRNA，即tRNAMetm。用胰核糖核酸酶A[EC 3.1.4.22]和核糖核酸酶T1[EC 3.1.4.8]消化纯化的tRNAMetm。将消化产物分离成单个组分并进行完全测序。两种核糖核酸酶消化产物的序列分析结果非常吻合，表明该tRNA的链长为76，包括13个修饰核苷酸。如随附论文所述，通过对核糖核酸酶T1部分消化产物的分析，假定tRNA具有三叶草叶结构的一些传统特征，这些寡核苷酸片段可以构建成一个独特的总序列。
