Control of cell-free protein synthesis by amino acids: effects on tRNA charging

In order to resolve the observation that addition of glutamine and glutamate appears to be of particular importance in enhancing the activity of a cell-free protein synthesis system derived from rat liver (Manchester and Tyobeka, 1980), we have measured the KM of the aminoacyl-tRNA synthetases towards amino acids and the extent of aminoacylation of tRNA under the conditions of our earlier experiments. During incubation of the cell-free system in the presence of an amino acid mixture the extent of acylation to tRNA of 15 amino acids studied showed no clear change from initial time values. When incubation took place in the absence of added amino acids, however, the levels of glutamate and glutamine bound to their appropriate tRNAs dropped more rapidly and to lower levels than for other amino acids except tryptophan. The pronounced drop for these two amino acids does not seem to result from an abnormally high KM value for the synthetases towards the respective amino acids, nor an abnormally low Vmax, but probably from the fact that the amounts of glutamyl and glutaminyl-tRNA in the cell-free system are comparatively low.