RNA-directed cell-free synthesis of the galactose enzymes of Escherichia coli

Summary Conditions are described for the RNA-directed cell-free synthesis of the three galactose enzymes of Escherichia coli. Together with the DNA-directed synthesis described previously, this system permits the measurement of the three gene-products encoded by the gal operon as active enzymes synthesized in vitro in response to either gal-DNA or gal-RNA. The yield of enzyme is proportional to the amount of RNA added. Thus, the RNA-directed enzyme synthesis can serve as an assay system for functional mRNA. This test has been employed to determine the kinetics of synthesis and degradation of functional gal mRNA under the conditions of cell-free enzyme synthesis. The functional half-life of gal mRNA in this system is 6–7 minutes and is higher than expected from in vivo measurements.In contrast to the DNA-directed cell-free synthesis, the RNA-directed synthesis of the galactose enzymes is neither stimulated by cyclic adenosine-3:5-monophosphate nor by inducer.