The LexA protein of E. coli is a repressor of at least 20 genes in the SOS regulon, and by this function plays a major role in regulating the SOS response. Two different genetic approaches have been taken to define the DNA binding domain of LexA repressor. First, several mutant repressors which are defective in DNA binding have been isolated. The mutations generating these repressors were dominant to lexA+, indicating that the mutant proteins can act in trans to interfere with binding of normal repressor to an operator sequence. The repressors may be defective due to elimination or disruption of contacts made between side chain(s) within the protein and the DNA helix but dominant because they can still interact with other monomers of LexA protein. In a second experiment to define the DNA binding domain of LexA protein, a novel genetic selection has been used to isolate DNA binding specificity mutants. The recA operator (CTG TATGA.GCATA CAG), a known lexA binding site, has been altered in a symmetric fashion. This choice was based on the assumption that the dyad symmetry of the operator indicates at least two repressor monomers bind to each operator such that each monomer recognizes one half of the operator. A class of mutant repressors which restored binding to this altered operator but had little affinity for the wild-type recA operator was isolated. This type of mutation allowed the identification of amino acids in the repressor which are likely to make specific contacts with base-pair(s) in the DNA binding site. By examining the effects of a series of amino acid substitutions on repressor specificity, it was possible to show that a glutamic acid residue at position 45 (E45) contacts the first and last base-pair of the consensus recA operator (CTG TATGA.GCATA CAG). Both negative dominant and operator recognition mutations were located in a small region that was previously identified to specify a helix-turn-helix motif based on sequence similarity to other repressors. These studies therefore suggest that LexA protein may bind to DNA by a helix-turn-helix motif similar to these repressors.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/184848 |
| Date | January 1989 |
| Creators | Thliveris, Andrew Tom. |
| Contributors | Mount, David W., Ito, Junetsu, Brower, Danny L., Bohnert, Hans J., Little, John W. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | English |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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