In vitro regulation of the Deo operon of E.coli at the initiation of transcription

Abstract

The formation of functional mRNA from the Deo operon of Escherichia coli has been investigated in a coupled and uncoupled protein synthesizing in vitro system using the antibiotics rifampicin, streptolydigen and chloramphenicol. Initiations of transcription take place from 1 to 10 min after the start of the incubation. Elongation of mRNA proceeds for about 20 min, while translation of mRNA into active enzyme occurs for at least 50 min. In the coupled system the half-life of thedeoA mRNA is about 3 min. Cell-free enzyme synthesis has been carried out by the use of phenol extracted mRNA synthesized either in the presence or absence of amino acids. The amount of enzyme formed is directly proportional to the amount of mRNA added and independent of whether the mRNA has been synthesized in the presence or absence of amino acids. By separating the transcription of thedeoA mRNA from its translation, it has been shown that the DeoR and CytR repressors as well as the cyclic AMP-catabolite activator protein control the initiation of transcription.