Internal promoter in the ilvGEDA transcription unit of Escherichia coli K-12.
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 beta-galactosidase. Using this method we have quantitatively evaluated in vivo (i) the activity of previously described promoter, pG, preceding ilvG; (ii) the relative activity of pE 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, delta(ilvGEDA), delta(ilvED), and ilvA538 hosts. The potential effects of these alleles that were previously postulated to affect ilvGEDA expression were assessed in vivo by monitoring beta-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 beta-galactosidase expression with ara-ilv-lac fusions paralleled the in vivo activity.
D001435:Bacteriophages / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D009154:Mutation; D009876:Operon; D010957:Plasmids; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D004269:DNA, Bacterial; D001616:beta-Galactosidase