Isolation of E. coli promoters from the late region of bacteriophage T7 DNA

Abstract

Promoter sequences recognized by Escherichia coli RNA polymerase have been isolated from bacteriophage T7 DNA using the plasmid pBRH4. T7 DNA was digested with the restriction endonucleases Hae III, Alu I, and Eco RI* and the products of these digestions were ligated into the Eco RI site of pBRH4. Cloning of Hae III and Alu I-digested T7 DNA was achieved by blunt-end ligation of these fragments to the polymerized ends of Eco RI-cleaved pBRH4. This converts blunt-end fragments of T7 DNA into cohesive-end Eco RI fragments. Promoter-containing T7 restriction fragments were selected by activation of the tetracycline-resistance gene located on the plasmid vector. The genomic location of each T7 insert was determined using the “Southern-blotting” technique and Hpa I-cleaved T7 DNA. Two promoter-active restriction fragments are thought to contain the C and E promoters of T7. However, the majority of the promoter-active fragments cloned map within the late gene region of T7. In vitro binding studies indicate that E. coli RNA polymerase can form heparin resistant complexes with the cloned T7 DNA promoter fragments. These results suggest that while E. coli RNA polymerase may not participate directly in the transcription of late T7 genes, promoters for this enzyme are present in this region of the DNA.