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  • Title: Spacing requirements between LexA operator half-sites can be relaxed by fusing the LexA DNA binding domain with some alternative dimerization domains.
    Author: Oertel-Buchheit P, Schmidt-Dörr T, Granger-Schnarr M, Schnarr M.
    Journal: J Mol Biol; 1993 Jan 05; 229(1):1-7. PubMed ID: 8421295.
    Abstract:
    The dimerization domain of the LexA repressor has been replaced by two heterologous dimerization motifs: the "leucine zipper" from the jun oncogene product and the carboxy-terminal oligomerization domain of Escherichia coli lac repressor. The corresponding hybrid proteins LexA1-87-Jun zipper and LexA1-87-lac repressor have been purified and their DNA binding properties have been studied using gel mobility shift assays. Both fusion proteins form stable specific complexes with a short DNA duplex harboring the CTGT(at)4ACAG consensus sequence of the LexA repressor. This conserved DNA binding capacity distinguishes these two fusion proteins from many others containing a LexA DNA binding domain fused to different heterologous transactivation and/or dimerization domains. However the fusion proteins LexA1-87-Jun zipper and LexA1-87-lac repressor behave differently from native LexA repressor in that these fusion proteins tolerate the insertion of additional base-pairs between the two invertedly repeated CTGT motifs. LexA1-87-Jun zipper requires two CTGT motifs and tolerates the insertion of at least two additional base-pairs between these motifs, whereas LexA1-87-lac repressor requires in fact only a single CTGT motif for the formation of a specific complex detectable in gel mobility shift assays. The inability of the normal LexA repressor to form well-defined complexes with operators containing additional base-pairs in the center suggests that the LexA "hinge region" between the amino-terminal DNA binding and the carboxy-terminal dimerization domain might not be entirely flexible. In an attempt to remove a hypothetical interaction between the LexA cleavage site (which is situated within the hinge region) and the catalytic cleavage center (situated within the carboxy-terminal domain) a LexA mutant repressor containing five simultaneous mutations in the hinge region has been constructed and purified. Surprisingly this mutant repressor failed to form stable complexes detectable by the gel mobility shift assay even with the normal consensus sequence, suggesting that the LexA hinge region is more than a simple connector between the two structural domains and that its chemical nature is important not only for LexA cleavage, but also for the formation of stable LexA-DNA complexes.
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