A study of the lac repressor hinge region was undertaken to decipher its role in the function of DNA binding and communication between binding sites within the protein. Specific mutations were designed, and the proteins purified from these constructs were used in various biochemical experiments. These mutants revealed alterations in DNA binding as well as in the response to inducer.
The first set of mutations involved insertion of glycines to the hinge region in an attempt to disrupt communication between the binding domains. The placement of the insertions was important for the maintenance of DNA binding. As the number of insertions increased, the protein's affinity for the natural operator decreased, approaching activity for non-specific DNA. Another mutation introduced within the hinge region involved the addition of a disulfide bond across partner hinges within the dimer of LacI. This alteration apparently stabilizes the N-termini to a significant degree and prevents operator dissociation in the presence of inducer for the oxidized protein. This mutant protein in both oxidized and reduced forms also demonstrated high affinity for the natural operator as well as several variant constructs.
These mutants offered the potential to study the effect of operator sequence on the protein's interaction with DNA. Variant operators were constructed that altered the spacing and/or the symmetry of the half-sites. A broad range of responses are reported, some of which were not predicted based on the wild-type operator binding or inducibility for the particular protein under examination.
In composite, the results reported in this thesis confirm a key role for sequence and structure in the hinge region of this protein in determining both affinity for operator DNA (and variants) and the allosteric response to inducer. Perhaps more importantly, however, the binding properties of these mutant lactose repressors with operator DNA variants illuminate a less well understood and appreciated phenomenon---the role of non-contacted DNA sequence and therefore presumably conformation in this regulatory interaction. These observations provide a strong base for detailed exploration of the mechanisms by which DNA sequence variation influences binding parameters for regulatory proteins.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/19378 |
| Date | January 1999 |
| Creators | Falcon, Catherine Margaret |
| Contributors | Matthews, Kathleen S. |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | 223 p., application/pdf |
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