The DNA-directed in vitro protein synthesizing system of Salmonella typhimurium: The effect of glutamate substitution

The effect of potassium glutamate was examined on the DNA-directed in vitro protein synthesizing system of Salmonella typhimurium which conventionally contained acetate as a sole counter anion. The glutamate replacement increased the potassium optimum by about 70% and improved the expression of different DNA templates, but selectively. The biggest improvements in expression (about 8-fold) were seen with a lacUV5 (from Escherichia coli) template and with a mutant promoter his operon (from S. typhimurium) template. In contrast, the expression of a leuV promoter (from Escherichia coli) template was relatively unaffected by the glutamate replacement. The chain-growth-rate of mRNA and polypeptide syntheses in the DNA-directed in vitro protein synthesizing system were unaffected by the glutamate replacement. It was concluded that at least a part of the effect of glutamate replacement is on RNA polymerase-promoter interaction, and most likely the association step. Glutamate replacement did not alter the ppGpp-mediated positive and negative regulation of the his and leuV promoter, respectively, in the in vitro system. Taken together, the results suggest that the use of potassium glutamate in place of potassium acetate in DNA-directed in vitro synthesis provides a physiologically more relevant approximation of the ionic environment in vivo.