Internal promoter in the ilvGEDA transcription unit of Escherichia coli K-12

Abstract

Segments of the ilvGEDA transcription unit have been cloned into the promoter tester plasmid pMC81. This vector contains cloning sites situated upstream of the lacZ gene coding for β-galactosidase. Using this method we have quantitatively evaluated in vivo (i) the activity of previously described promoter, p(G), preceding ilvG; (ii) the relative activity of p(E) promoter, previously postulated to be located between ilvG and ilvE; and (iii) the effect of the frameshift site present in the wild-type ilvG gene by comparison with mutant derivatives lacking this frameshift site. Isogenic derivatives of strain MC1000 were constructed by transduction with phage P1 grown on rho-120, Δ(ilvGEDA), Δ(ilvED), and ilvA538 hosts. The potential effects of these alleles that were previously postulated to affect ilvGEDA expression were assessed in vivo by monitoring β-galactosidase production directed by ilv DNA fragments. Cloned ilv segments were also tested for activity in vitro with a DNA-directed coupled transcription and translation system. The production in vitro of ilv-directed ilv gene expression and β-galactosidase expression with ara-ilv-lac fusions paralleled the in vivo activity.