In vitro analysis of the Escherichia coli RNA polymerase interaction with wild-type and mutant lactose promoters

Abstract

Two methods have been used to study the interaction of Escherichia coli RNA polymerase with the wild-type E. coli lactose promoter ( lacP) and with various lacP mutants. Using a nitrocellulose filter binding assay, we have compared the association kinetics of in vitro heparin-resistant RNA polymerase- lacP complexes formed with the wild-type promoter (as represented by a lacO- Z deletion) and several catabolite gene activator protein(CAP)-independent mutant promoters. Analysis of six mutants has shown that the more lac expression is relieved from catabolite repression in vivo, the faster lacP saturates with RNA polymerase in vitro. A transcriptional system directed by purified lacP-containing restriction fragments has provided a more quantitative analysis of the binding reaction. Whether in the presence of CAP, 25% glycerol or in the absence of a positive affector, promoters whose expression is less dependent on CAP saturate faster with RNA polymerase and to higher levels. The L157 lac promoter mutation, which is thought to inhibit a functional RNA polymerase-promoter interaction, has been examined. Although RNA polymerase binds the L157 mutant promoter fragment in a filter-retainable complex to levels slightly higher than those attained by the wild-type lac promoter, transcriptional studies have revealed that such complexes are unable to efficiently initiate RNA synthesis. CAP and 25% glycerol have no appreciable effect on the initiation frequency. These methods have also been used to determine the extent of the promoter sequence that is required for the formation of a stable and functional RNA polymerase-DNA complex, lacP which has been endonucleolytically restricted to different sizes as well as a novel ligation product of endonucleolytically destroyed promoter fragments has been examined for the ability to interact with RNA polymerase. A sequence specificity upstream from the semi-conserved RNA polymerase binding heptanucleotide is found to be necessary for optimal utilization of the lac promoter.