Initiator methionine residues at the NH2-termini of the two precursors of MOPC-41 immunoglobulin light chain. Studies with the initiator and internal tRNAMet species.

Abstract

The mRNA molecules coding for the MOPC-41 immunoglobulin light chain direct the cell-free synthesis of two precurosors in which 22 or 20 amino acid residues precede the NH2-terminus of the mature light chain. The two NH2-terminal residues of the long extra piece (22 residues) are missing in the shorter extra piece (20 residues), in the other 20 positions both extra pieces have an identical sequence. The sequences are: in the long extra piece, Met-Asp-Met-Arg-Ala-…, in the short extra piece, Met-Arg-Ala-… [Burstein, Y. and Schechter, I. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 716–720]. To study the relation between these molecules we analysed the amino acid sequence of precursors synthesized in the presence of the initiator [35S]Met-tRNA1Met or internal [35S]Met-tRNA2Met as sole source of label. The results show that the initiator [35S]Met-tRNA1Met inserts [35S]methionine only at the NH2-terminal position; the internal [35S]Met-tRNA2Met does not label Met-1 but inserts [35S]-methionine inside the polypeptide chain at correct positions. These findings demonstrate that the NH2-terminal methionine, present in both precurosors, is the initiator residue. Therefore, the two precursors are the direct product of mRNA translation, and the short precursor does not originate from the longer precursor by cleavage of the Met-Asp dipeptide of the long extra piece. The identification of initiator methionine strengthens the contention that the precursor is synthesized intracellularly. Sequencing of precursors labelled with either [35S]Met-tRNAMet species yielded discrete radioactive peaks of NH2-terminal or internal methionines, without cross-contamination. These results provide positive and controlled characterization of both the initiator and internal tRNAMet species, using a natural mRNA. The possibility of two initiation sites for translation on a single mRNA molecule is discussed.