Unusual properties of promoter-up mutations in the Escherichia coli galactose operon and evidence suggesting RNA polymerase-induced DNA bending

Abstract

Two mutations are described, each of which renders the Pribnow box sequence of one of the two overlapping promoters of the Escherichia coli galactose operon identical to the consensus sequence TATAAT. Both double exchanges were specifically introduced into the original context by oligonucleotide-directed mutation construction. Each of the mutant promoters exhibits a greatly enhanced capacity to form stable complexes with RNA polymerase, as judged by nuclease protection experiments and by assaying shifts of electrophoretic mobility. On the other hand, the effect of the same mutations on the rates of transcription from the two gal promoters is strikingly different. Unexpectedly, when complexed with RNA polymerase, DNA fragments carrying one of the two double exchanges were found to differ from each other as well as from the corresponding wild-type fragment with respect to their electrophoretic mobilities. These observations are indicative of different three-dimensional structures of these complexes which may reflect different forms of DNA bending induced in these otherwise identical fragments by complex formation with RNA polymerase.