Influence of a protein synthesis inhibitor from barley seeds upon different steps of animal cell-free protein synthesis

Abstract

The mechanism of action on animal cell-free protein synthesis by the translation inhibitor II from barley seeds has been studied using the rabbit reticulocyte lysate protein synthesis system. No influence was detected on the ribosomal binding of initiator-tRNA, met-tRNAf, in the presence of barley hordein mRNA, or on the aminoacyl-tRNA synthetase catalyzed formation of aminoacyl-tRNA, suggesting the inhibitor to be without effect both on the protein synthesis initiation and on the supply of aminoacyl-tRNAs. Inhibition of reactions in the elongation cycle was examined by measurement of the transfer of the nascent peptide from peptidyl-tRNA to puromycin. The observed lack of influence on the puromycin reaction implies that the barley inhibitor neither blocked the peptidyltransferase nor the translocation activities. In addition, the high amount of Phe-tRNA bound to ribosomes in the presence of inhibitor and poly(U) suggested the aminoacyl-tRNA binding step to be unaffected by the inhibitor. These findings are compatible with inhibition of a reaction in the elongation cycle subsequent to the aminoacyl-tRNA binding but preceding the translocation.