Construction and characterization of E. coli promoterprobe plasmid vectors II. RNA polymerase binding studies on antibiotic-resistance promoters

The binding of Escherichia coli RNA polymerase to antibiotic-resistance promoters was examined using the nitrocellulose filter assay. Four filter-retainable HaeIII fragments were observed with pBR322 and the promoter-probe plasmids, pBRH1, pBRH2 and pBRH4. Of the three fragments studied, two were shown to carry promoters for the ampicillin (Ap) and tetracycline (Tc) resistance genes, while the third present in pBRH1 appears to be the promoter for colicin E1 immunity (Col^imm). Although the formation of filter-retainable complexes involving the Tc^r promoter was sensitive to high salt, Ap^r promoter complexes were not. It was also shown that plasmids containing only the "firm-binding" portion of the Tc^r promoter crould still bind RNA polymerase in vitro despite the fact that these plasmids confer no in vivo Tc^r. Additional filter-binding experiments performed with AluI-digested pBR322 DNA revealed the presence of a fifth RNA polymerase binding site on pBR322. This site is probably the promoter for the 100 bp transcript thought to be involved in the initiation of plasmid replication. An analysis of the recombinant plasmid (pKTR25) which carries the Kan-B portion of the EcoRI kanamycin (Kn) resistance fragment revealed that this fragment contains two RNA polymerase binding sites. We believe that these sites are responsible for the insertional activation of the Tc^r gene and may be the promoters for the Kn^r and fusidic acid (Fa) resistance genes.