Studies on Polynucleotides: XCI. Yeast Methionine Transfer Ribonucleic Acid: Purification, Properties, and Terminal Nucleotide Sequences

Abstract

Abstract Countercurrent distribution of baker's yeast transfer RNA (tRNA) gives rise to two peaks (I and II) of methionine acceptor activity. Methionine tRNA (tRNAfmet) (Peak I) has been purified to homogeneity by ion exchange chromatography on DEAE-Sephadex and by reverse phase chromatography. It has the following properties. (a) It can be aminoacylated with methionine by either crude yeast or Escherichia coli aminoacyl tRNA synthetases. (b) The methionyl-tRNA thus formed can be further converted to N-formyl-methionyl-tRNA (fMet-tRNAfmet) in the presence of formyl tetrahydrofolic acid and E. coli methionyl-tRNA transformylase. (c) Amino acid incorporation studies in vitro with poly r(U-G) and poly r(A-U-G) as messengers show that, similar to E. coli fMet-tRNAfmet, the yeast fMet-tRNAfmet can bring about the initiation of protein synthesis under the direction of either of the initiator codons G-U-G or A-U-G. Analysis of fragments produced by the action of takadiastase Ribonuclease T1 show that the 5'- and 3'-terminal nucleotide sequences of yeast tRNAfmet are pA-G-, and C-U-A-C-C-A, respectively; the terminal sequences thus derived are not identical with those present in E. coli tRNAfmet. Under conditions of magnesium ion concentration (5 mm) used for the aminoacylation of tRNAfmet (Peak I), tRNAmet (Peak II) is aminoacylated by crude E. coli aminoacyl tRNA synthetases at a slow rate. At 15 mm Mg++, however, tRNAmet (Peak II) is aminoacylated with methionine to the same extent by crude E. coli aminoacyl tRNA synthetases as by yeast aminoacyl tRNA synthetases. Met-tRNA from Peak II cannot be converted to fMet-tRNA by E. coli extracts.