The effects of deacylated transfer ribonucleic acid on polypeptide-chain initiation in Ehrlich-ascites-tumour-cell extracts [proceedings

Abstract

In intact Ehrlich ascites-tumour cells the rate of protein synthesis is decreased by over 6096 in the absence of a single essential amino acid (van Venrooij et al., 1972). This effect is due largely to an inhibition of polypeptide-chain initiation, and is associated with polyribosome disaggregation and a decrease in the number of Met-tRNAme-40 S subunit initiation complexes (Pain & Henshaw, 1975). Cell-free extracts prepared from control and lysinedeprived cells show some of the characteristics of the cells from which they were derived. Protein synthesis in starved-cell extracts is decreased by up to 75% relative to that in fed-cell extracts. Starved-cell extracts also have a diminished ability to form initiation complexes between 40s subunits and Met-tRNAme. This defect is abolished by addition of highly purified initiation factor eIF-2 (Pain et al., 1980; Clemens et al., 1980). It has been suggested (Vaughan & Hansen, 1973; Kyner el al., 1973) that the inhibition of polypeptide-chain initiation seen in cells deprived of amino acids may be due to increases in the concentrations of deacylated tRNA species. To test whether. increased concentrations of deacylated tRNA could directly inhibit polypeptide-chain initiation, the effect of adding periodate-oxidized tRNA (which cannot be charged with amino acids) to cell-free extracts from Ehrlich ascites-tumour cells was examined. Cell-free extracts from Ehrlich ascites-tumour cells were prepared, and analysis of labelled initiation complexes by sucrose-gradient centrifugation was performed as described by Pain et al. (1980). Deacylated calf liver tRNA was subjected to periodate oxidation as described by Vaughan & Hansen (1973). Addition of deacylated oxidized tRNA to cell-free extracts From Ehrlich ascites-tumour cells in the presence of 1 mMemetine to prevent polypeptide-chain elongation causes only a 10-2096 decrease in the concentration of Met-tRNAmet-40 S subunit initiabon complexes, at concentrations between 20 and 1OOpg of added tRNA/ml (Table 1). This small inhibition is not concentration-dependent. A much greater effect is seen on Met-tRNAme-80S ribosome initiation-complex formation. Addition of 20pg of deacylated tRNA/ml causes an 85% decrease in the concentration of Met-tRNAMet-80 S ribosome complexes. At higher concentrations of added tKNA Met-tRNAmet-80S ribosome complexes are completely abolished (Table 1). Addition of deacylated oxidized tRNA to cell-free extracts in the absence of emetine, where protein synthesis is occurring, causes an accumulation of Met-tRNAme-40S subunit initiation complexes. This implies impairment of a stage after MettRNAWCt-40S subunit initiation-complex formation, possibly 40s subunit-60s subunit joining. However, h this situation the concentrations of complexes seen just reflect the balance between their rate of formation and their rate of utilization, and the results are more difficult to interpret than those of experiments performed in the presence of an inhibitor of elongation. We have shown that concentrations of deacylated oxidized tRNA in the range 20-100pg/ml cause only a small decrease in the Concentration of Met-tRNAwd-40S subunit initiationcomplex formation. Since the concentration of Met-tRNAme40s subunit initiation complexes in extracts from lysinedeprived cells is decreased by up to 77% compared with that in extracts from control cells (Pain et al., 1980; Clemens et al., 1980), the small decline in initiation complexes seen with deacylated tRNA is not enough to account for the differences between fed-cell and starved-cell systems. It would seem therefore that an increase in the concentration of deacylated tRNA after amino acid deprivation is not the direct cause of the inhibition of this step of polypeptide-chain initiation in Ehrlich ascitestumour cells under these circumstances. Deacylated tRNA is strongly inhibitory to Met-tRNAmet-80s ribosome initiation-complex formation. This effect may be due to impairment of the conversion of Met-tRNAm-40S subunit initiation complexes into Met-tRNAmet-80S ribosome initiation complexes or to a destabilization of the latter complexes. The accumulation of Met-tRNAmet40S subunit initiation complexes seen in the presence of deacylated tRNA under protein-synthesis conditions is consistent with the former model. These results are also in agreement with the findings obtained by Kyner et al. (1973), who reported an inhibition of Met-tRNAmd-80S ribosome initiation-complex formation by uncharged tRNA in a fractionated cell-free system. As deacylated tRNA is unlikely to be directly responsible for the inhibition of polypeptide-chain initiation at the stage of Met-tRNAme40s subunit initiation-complex formation, the mechanism by which this part of the protein-synthesizing machinery of Ehrlich ascites-tumour cells recognizes the lack of one amino acid remains to be elucidated. This work is supported by grants from the Medical Research Council and the Cancer Research campaign. Clemens, M. J., Henshaw, E. C. & Pain, V. M. (1980) Blochem. Soc. Trans. 8,350-351