A new class of promoter mutations in the lactose operon of Escherichia coli

The isolation and genetic characterization of a number of mutations that are located in the promoter region of the lac2 operon are described. These mutations have reduced levels of lac operon expression in a wild, type (crp⁺cya⁺) genetic background. Three of the mutations also have lower levels of lac operon expression than lacP⁺ in a crp^?cya^? genetic background, that is in the absence of the catabolite activator protein and 3′,5′-adenosine cyclic monophosphate. These three mutations are located nearest to the lac operator. They define a second essential site in the promoter region.     

Recognition sequences of repressor and polymerase in the operators of bacteriophage lambda

Nucleotide sequences in two wild-type and six mutant operators in the DNA of phage λ are compared. Strikingly similar 17 base pair units are found which we identify as the repressor binding sites. Each operator contains multiple repressor binding sites separated by A-T rich spacers. Elements of 2 fold rotational symmetry are present in each of the sites. Superimposed on each operator is an E. coli RNA polymerase recognition site (promoter). Similarities in the sequences of the two λ promoters, a lac promoter, and an E. coli RNA polymerase recognition site in SV40 DNA are noted.     

Mechanism of promoter repression by Lac repressor–DNA loops

The Escherichia coli lactose (lac) operon encodes the first genetic switch to be discovered, and lac remains a paradigm for studying negative and positive control of gene expression. Negative control is believed to involve competition of RNA polymerase and Lac repressor for overlapping binding sites. Contributions to the local Lac repressor concentration come from free repressor and repressor delivered to the operator from remote auxiliary operators by DNA looping. Long-standing questions persist concerning the actual role of DNA looping in the mechanism of promoter repression. Here, we use experiments in living bacteria to resolve four of these questions. We show that the distance dependence of repression enhancement is comparable for upstream and downstream auxiliary operators, confirming the hypothesis that repressor concentration increase is the principal mechanism of repression loops. We find that as few as four turns of DNA can be constrained in a stable loop by Lac repressor. We show that RNA polymerase is not trapped at repressed promoters. Finally, we show that constraining a promoter in a tight DNA loop is sufficient for repression even when promoter and operator do not overlap.     

On the Mechanism of Action of <Emphasis Type="Italic">lac</Emphasis> Repressor

Chen et al. have proved conclusively that lac repressor and RNA polymerase bind independently to wild type lac DNA in vitro. To explain the lacp ^s mutation, which causes competitive binding between repressor and polymerase, they suggest that a new promoter site has been created near the lac operator.     

Regulation of the regulatory gene for the arabinose pathway,araC

The promoter of the araC gene was fused to the structural genes of the lac operon using the techniques described in the preceding paper. The resulting fusion strains were used to study the regulation of the araC gene by assaying the fused lac gene products. It was found that the expression of the fused lac genes was repressed by the product of the araC gene and was regulated by the cyclic AMP catabolite control system. This implies that the araC gene itself is repressed by its own product and is catabolite regulated. These findings introduce a new level of complexity in the regulation of the arabinose pathway of Escherichia coli.     

Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the -35 to -10 spacing

A series of promoter hybrids has been constructed by exchanging the ? 35 and ? 10 regions of lacUV5, tet, and trp promoters. These three promoters and the six hybrid promoters constructed from them have been inserted into a pKO plasmid which places galactokinase expression under the control of the inserted promoter. Additionally, promoter mutants were prepared which had altered the spacing between the ? 35 and ? 10 regions of the promoter. Derivatives of the tet promoter with one or two extra base pairs in this spacer region and constructions of the lac:: tet hybrid promoter with two different spacings have been inserted into the galactokinase expression plasmid. Measurements of galactokinase levels in strains harboring these plasmids permited the comparison of in vivo activities of the promoters. The strongest of the hybrid promoters (order: ? 35, ? 10) were trp:: lac and trp:: tet suggesting a high efficiency for the ? 35 region of the trp promoter. The weakest promoters were tet:: trp, lac:: trp and lac::tet indicating a weak ? 10 region for the trp promoter and the importance of ? 35 to ? 10 spacing. Analysis of activity of related promoters with differences in spacing indicated that a distance of 19 bp yields a very weak promoter, and that 18 bp is less active than the 17-bp spacing, which is the most frequently found spacing in promoters.