In vivo experiments on the mechanism of action of L-arabinose C gene activator and lactose repressor.

Simultaneous exposure of the cell interior of a rifampin-permeable mutant, Ewherichia coli to inducer and rifampin demonstrated that 8% &3% of the ara operon DNA copies and 1% f 3% of the Zac DNA copies contain an RNA polymerase molecule insensitive to rifampin inhibition. Although previous genetic studies have shown that the operons are controlled via different mechanisms, the data presented here show that both operons are controlled in such a way that an uninduced operon copy has a low probability of possessing an RNA polymeraee molecule that has initiated transcription. These in tivo measurements were made possible by the development of techniques to determine accurately the times required for rifampin and inducers to diffuse into cells and attain effective concentrations. 100 pg rifampin/ml requires 4-4 seconds to enter and block further initiations by RNA polymerase, whereas fully inducing concentrations of L-arabinose and IPTGt are effective in less than one second. The initial rifampin entry rate into cells is proportional to its extracellular concentration. This relationship allowed calculation of the enzyme induction that would have been observed if the rifampin entry time were zero.