Role of DNA regions flanking the tryptophan promoter of Escherichia coli. II. Insertion of lac operator fragments.

To examine the effect of introducing protein-binding sites around a promoter upon expression from that promoter, a series of altered tryptophan promoter plasmids have been constructed. In these derivatives of pKO-1 (a galK-expression, promoter-assay vector), the trp promoter has been introduced into the vector, such that the galK expression is dependent on the trp promoter. Unique restriction sites have been introduced adjacent to the trp promoter for the insertion of other DNA fragments. The DNA-binding site (a lac operator (lacO) fragment of 33 bp) for the lactose repressor was inserted into the trp promoter-pKO plasmids at various defined locations from -52 to +58 relative to the start site of transcription. Strains bearing tryptophan promoter-lactose operator plasmid derivatives were assayed for galactokinase activity in Escherichia coli C600 and JM103 (a lacIQ strain) to observe the effect of lac repressor binding and removal. In those plasmids where the lac repressor was downstream from the promoter, expression was diminished by the presence of the lactose repressor and galactokinase activity could be induced by addition of IPTG. An analogous lac promoter plasmid was repressed over 90%, and the plasmid containing the lacO fragment at +2 exhibited up to 80% repression; 40-50% repression was observed when the lacO was placed at positions +27 and +58. Placement of the lacO at the upstream locations -39 and -52 produced lower repression. To examine the effects of more than one operator surrounding the promoter, plasmids were constructed that had a lacO at -39 or -52 and, in addition, had an operator downstream.+2