This report describes an Escherichia coli genetic system that permits bacterial genetic methods to be applied to the study of essentially any prokaryotic or eukaryotic site-specific DNA binding protein. It consists of two parts. The first part is a set of tools that facilitate construction of customized E.coli strains bearing single copy lacZYA reporters that are repressed by a specific target protein. The second part is a pair of regulatable protein expression vectors that permit in vivo production of the target protein at levels appropriate for genetic experiments. When expressed in a properly designed reporter strain, the target protein represses the lac genes, resulting in an E.coli phenotype that can be quantitatively measured or exploited in large scale genetic screens or selections. As a result, large plasmid-based libraries of protein genes or pools of mutagenized variants of a given gene may be examined in relatively simple genetic experiments. The strain construction technique is also useful for generating E.coli strains bearing reporters for other types of genetic systems, including repression-based and activation-based systems in which chimeric proteins are used to examine interactions between foreign protein domains.

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The related phage k and phage P22 repressors each bind cooperatively to adjacent and separated operator sites, an interaction that involves a pair of repressor dimers. The specificities of these interactions differ: Each dimer interacts with its own type but not with dimers of the heterologous repressor. The two repressors exhibit significant amino acid sequence homology in their carboxy-terminal domains, which are responsible for both dimer formation and the dimer-dimer interaction. Here, we identify a collection of amino acid substitutions that disrupt the protein-protein interaction of DNA-bound k repressor dimers and show that several of these substitutions have the same effect when introduced at the corresponding positions of P22 repressor. We use this information to construct a variant of the k repressor bearing only six non-wild-type amino acids that has a switched specificity; that is, it binds cooperatively with P22 repressor, but not with wild-type k repressor. These results identify a series of residues that determine the specificities of the two interactions.

The Bacillus subtilis veg promotor site was shown to be composed of two neighboring RNA polymerase binding sites, only one of which ("Site I") produces a transcript. The relationship between these sites has been investigated following insertion of ten base pairs of DNA between the sites and subsequent cloning of each site independent of the other, and deletion of DNA sequences upstream from the productive site. The effect of DNA insertion was to permit transcription initiation from the previously nonproductive Site II, in either the presence or absence of Site I. This could be attributed to insertion of a purine residue seven base pairs downstream from the Pribnow box of Site II. Transcription from Site I was slightly increased in the presence of Site II in cis, but was still efficient in the absence of Site II, indicating that there is no absolute dependence on Site II for in vitro transcription from Site I.