Preparation and analysis of l-[ 35S]methionine labeled transfer ribonucleic acids from rabbit liver

Abstract

The two methionine-specific tRNA species found in rabbit liver cytoplasm can be individually labeled with radioactive methionine under the proper conditions. Crude rabbit liver tRNA aminoacylated with radio-active methionine using the homologous rabbit methionyl-tRNA synthetase, and then deacylated using the Escherichia coli methionyl-tRNA synthetase, yields specifically labeled Met-tRNA m Met . This is the species of methionine-specific tRNA which transfers its methionine residue into internal positions in growing polypeptide chains. Transfer RNA f Met can be specifically aminoacylated using the E. coli methionyl-tRNA synthetase in the absence of a formyl donor to yield Met-tRNA f Met , the initiator tRNA in eukaryotes. In the presence of Leucovorin, a formyl group donor, the use of the E. coli methionyl-tRNA synthetase and the E. coli transformylase yields N-formyl-Met-tRNA f Met . This last tRNA species is not found naturally in the cytoplasm of eukaryotes, but it often can serve as a useful substrate in in vitro studies on the initiation of protein synthesis in eukaryotes. These labeling methods appear to be of general use for the methionine-specific tRNAs found in most eukaryotes.