The Primary Structure of Non-initiator Methionine Transfer Ribonucleic Acid from Bakers' Yeast

I. Purification and Complete Digestion with Ribonuclease T₁ and Pancreatic Ribonuclease A

抄録

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, tRNA^Met_m, was highly purified from the peak II fraction by successive chromatographic procedures. The purified tRNAmet 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, as in the cases of other tRNA's so far sequenced. The complete sequence of this tRNA was established by analyses of partial digestion products with RNase T₁, as reported in the accompanying paper.