The isolation and the characterization of DNA region bound to Escherichia coli RNA polymerase

Abstract

1. A method was established whereby after deoxyribonuclease digestion of an RNA polymerase-DNA complex, the DNA bound to the RNA polymerase was resistant to hydrolysis. 2. The amount of the resistant fraction was proportional to the amount of RNA polymerase added and reached saturation with increasing RNA polymerase. The saturation of the resistant fraction was 3% of the Escherichia coli DNA. 3. The resistant fraction of DNA sedimented sharply in coincidence with the RNA polymerase fractions having 22-S and 15-S sedimentation coefficients. Thus the fraction seemed to be part of an RNA polymerase-DNA complex. 4. Upon addition of substrates for RNA synthesis during the deoxyribonuclease digestion, such resistant DNA moieties disappeared. 5. The bound region of DNA was isolated free from RNA polymerase after deproteinization with phenol. The DNA moieties isolated from 22-S and 15-S complexes sedimented at 2.7 S and 2.4 S, respectively. 2.7-S DNA corresponds to 38 nucleotide pairs (130 Å), consistent with the dimensions of the 22-S RNA polymerase molecule previously proposed. 6. Reassociation of the isolated DNA moieties with 22-S and 15-S RNA polymerase exclusively was observed. This implies that each type of enzyme recognizes its specific site. 7. Chemical differences between 2.7-S and 2.4-S DNA moieties were established by base ratio analysis.