Initiator Methionine Transfer Ribonucleic Acid from Wheat Embryo: PURIFICATION, PROPERTIES, AND PARTIAL NUCLEOTIDE SEQUENCES

Abstract

Abstract Initiator methionine tRNA (tRNA1Met) species from wheat embryo has been purified by fractionation on a benzoylated DEAE-cellulose column followed by chromatography on a DEAE-Sephadex column. Transfer RNA1Met can be aminoacylated by the homologous enzyme as well as by Escherichia coli aminoacyl-tRNA synthetase. Unlike other eukaryotic cytoplasmic initiator tRNAs, the wheat embryo initiator tRNA cannot be formylated by E. coli transformylase. Amino acid incorporation studies in vitro using a wheat embryo ribosomal system and poly[r(A-U-G)] as messenger show that at low Mg2+ concentration polymethionine synthesis from Met-tRNA2Met (chain propagator species) is dependent on the presence of Met-tRNA1Met. Met-tRNA1Met cannot insert methionine internally in the homologous system but in a heterologous E. coli or rabbit reticulocyte ribosomal system Met-tRNA1Met appears to donate methionine into internal positions of the polypeptide chain. Analysis of oligonucleotides produced by digestion of tRNA1Met with ribonuclease T1 shows that the 5'- and 3'-terminal nucleotide sequences are pA-U-C-A-G- and A-U-A-C-A-Aoh, respectively. As in other eukaryotic cytoplasmic initiator tRNAs the tetranucleotide sequence T-ψ-C-G(A)- always present in loop IV of other tRNAs is absent. The tentative sequence of loop IV in the case of wheat embryo tRNA1Met is A-U*-C-G-m1A-A-A. Except for the presence of U* instead of U, this sequence is identical with those found in yeast, mouse myeloma, rabbit liver, and sheep mammary gland initiator tRNAs and different from that of E. coli initiator tRNA.