Lac repressor

Abstract: A method for studying the sequence-specific binding of proteins to DBA is described. The technique is a simple conjoining of the Maxam-Gilbert DNA-sequencing method and the technique of DNAase-protected fragment isolation. Fragments of a 5' end-labelled, double-stranded DNA segment, partially degraded by DNAase in the presence and absence of the binding protein, are visualized by electrophoresis and autoradiography alongside the base-specific reaction products of the Maxam-Gilbert sequencing method. It is then possible to see the protective "footprint" of the binding protein on the DNA sequence. The binding of lac repressor to lac operator is visualized by "footprinting" as an example. Equillibrium estimates indicate that 10-fold sequence-specificity (differential binding constant) could be studied easily using this technique.

Abstract: The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified. This gel method is applied to the study of the E. coli lactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promotor, but not with a CAP-insensitive mutant. Formation of a stable "open" or "melted-in" complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.

Abstract: Two hybrid promoters that are functional in Escherichia coli have been constructed. These hybrid promoters, tacI and tacII, were derived from sequences of the trp and the lac UV5 promoters. In the first hybrid promoter (tacI), the DNA upstream of position -20 with respect to the transcriptional start site was derived from the trp promoter. The DNA downstream of position -20 was derived from the lac UV5 promoter. In the second hybrid promoter (tacII), the DNA upstream of position -11 at the Hpa I site within the Pribnow box was derived from the trp promoter. The DNA downstream of position -11 is a 46-base-pair synthetic DNA fragment that specifies part of the hybrid Pribnow box and the entire lac operator. It also specifies a Shine-Dalgarno sequence flanked by two unique restriction sites (portable Shine-Dalgarno sequence). The tacI and the tacII promoters respectively direct transcription approximately 11 and 7 times more efficiently than the derepressed parental lac UV5 promoter and approximately 3 and 2 times more efficiently than the trp promoter in the absence of the trp repressor. Both hybrid promoters can be repressed by the lac repressor and both can be derepressed with isopropyl beta-D-thiogalactoside. Consequently, these hybrid promoters are useful for the controlled expression of foreign genes at high levels in E. coli. In contrast to the trp and the lac UV5 promoters, the tacI promoter has not only a consensus -35 sequence but also a consensus Pribnow box sequence. This may explain the higher efficiency of this hybrid promoter with respect to either one of the parental promoters.