Nucleoside diphosphate regulation of overall rates of protein biosynthesis acting at the level of initiation

A sensitive assay method developed to examine the effects of subtle, physiologically relevant, changes in the levels of adenine and guanine mono-, di-, and triphosphorylated nucleotides specifically on the initiation of protein synthesis is described. Initiation rates are quantified by measuring the amount of protein synthesis resulting from the run-off of ribosomes which have initiated during defined intervals in a modified in vitro protein-synthesizing system developed from Ehrlich ascites tumor cell lysates (Henshaw, E.C., and Panniers, R. (1983) Methods Enzymol. 101, 616-629). The modifications include the attenuation of the ATP-regenerating system so that the relative nucleotide levels more nearly reflect actual intracellular conditions. With this system the rate of initiation is highly sensitive to changes in the ADP:ATP and GDP:GTP ratios, but indifferent to the absolute levels of either diphosphate. While the tight coupling of these two ratios by endogenous nucleoside diphosphate kinase activity prevents the independent manipulation of either ratio, the data do eliminate both AMP and GMP per se as inhibitory species. The close agreement of our data calculated in terms of energy charge to previously published results on overall rates of protein synthesis in rat thymocytes (Mendelsohn, S.K., Nordeen, S.K., and Young, D.A. (1977) Biochem. Biophys. Res. Commun. 79, 53-60) continues to suggest a physiologically relevant regulatory influence of subtle changes in nucleotides acting at the level of the initiation reaction.