Utilization of Initiator Transfer Ribonucleic Acid Modified by Carcinogens Cholesteryl 14-Methylhexadecanoate and N-Methyl-N-Nitrosourea for Cell-Free Protein Synthesis

Abstract

(3H)Cholesteryl 14-methylehexadecanoate becomes bound to initiator tRNA from rat liver if incubated with it at 37°C. The binding of the ester was competitively inhibited by N-methyl-N-nitrosourea which has been previously demonstrated to alkylate guanine. It thus appears that the lipid reacts with guanosine residues in the molecule of tRNAF. Initiator tRNA preparations pretreated with cholesteryl 14-methylhexadecanoate were charged significantly better with L-methionine in the presence of aminoacyl-tRNA synthetases than comparable control preparations. The effect of the ester was in this respect comparable with that of N-methyl-N-nitrosourea. Incubation of (35S) Met-tRNAF modified by the lipid or N-methyl-N-nitrosourea with partially purified eIF-2 resulted in an enhanced formation of the ternary complex of this protein-synthesis factor. Also the sythesis of the 80S initiation complex was stimulated in the presence of these modified Met-tRNAs. Protein synthesis in a system composed of rabbit reticulocyte polyribosomes and partially purified initiation and elongation factors was stimulated if unfractionated aminoacyl-tRNA from rat liver pretreated with cholesteryl 14-methylhexadecanoate of N-methyl-N-nitrosourea was used. No changes were found in poly(U)-dependent peptide elongation when using aminoacyl-tRNA modified by either of these agents. Evidence was presented previously that cholesteryl 14-methylhexadecanoate is involved in the control of protein synthesis by affecting the binding sites of protein-synthesis factors and ribosomes for aminoacyl-tRNA, and protein phosphorylation. The present results suggest that the modification of initiator tRNA by this lipid may represent another pathway for its modulation of gene translation.