Investigation of the role of uncharged tRNA in the regulation of polypeptide chain initiation by amino acid starvation in cultured mammalian cells; a reappraisal.

Abstract

The rate of initiation of protein synthesis in mammalian cells in culture is regulated by the availability of essential amino acids. Previous reports have suggested that this effect is mediated by changes in the ratio of uncharged to charged tRNA species in these cells. We have investigated this possibility by examining the formation of 40‐S subunit. Met‐tRNAf ribosomal initiation complexes in extracts of Ehrlich ascites tumour cells; this is the stage in initiation previously shown to be regulated in response to lysine starvation of the intact cells. Exposure of cells to a low concentration of cycloheximide makes polypeptide chain elongation the ratelimiting step, allows reformation of polysomes in starved cells and abolishes the effect of lysine deprivation on overall protein synthesis. Treatment with a high concentration of cycloheximide inhibits all protein synthesis and ‘freezes’ the polysomes in the state of aggregation which exists in the cell at that moment. The latter conditions also permit the tRNA pool to become fully charged in both fed and lysine‐starved cells. In cell‐free extracts prepared from cells treated with either low or high concentrations of cyclolieximide 40‐S initiation complex formation nevertheless remains defective after lysine starvation of the cells. Addition of deacylated tRNA, oxidized with periodate to prevent its recharging, to initiating cell‐free extracts inhibits neither 40‐S initiation complex formation nor overall protein synthesis in vitro. Addition of lysine to an extract from lysine‐starved cells, under conditions where this amino acid is charged on to endogenous tRNA, fails to stimulate either 40‐S initiation complex formation or protein synthesis in vitro. These results suggest that amino acid starvation regulates the formation of 40‐S polypeptide chain initiation complexes in mammalian cells by a mechanism which is not influenced by the rate of protein synthesis and does not directly involve uncharged tRNA.