As the classical model for negative transcriptional control in prokaryotes and the subject of concentrated experimental attention, the lactose operon of Escherichia coli presents a well-defined system for studying genetic control through protein-DNA binding interactions. Binding of repressor at its cognate operator sequence within the regulatory region of the operon, while responsive to environmental conditions, efficiently inhibits transcription initiation by RNA polymerase. The high binding affinity and degree of specificity exhibited by this protein-DNA complex has encouraged investigation of the nature of the contacts formed.
We have explored specific contacts between the lac repressor and operator using 5-bromodeoxyuridine-substituted DNA. Substitution of BrdU for single thymidine positions in a synthetic 40 bp operator provides an indirect means of probing the major groove of operator DNA for critical contacts between the repressors and the 5-methyl of individual thymidines. As a photoreactive species, BrdU provides substrate for ultraviolet irradiation. Upon irradiation, strand scission occurs at the BrdU residues. When bound, lac repressor protein provides protection against UV-induced breakage depending on the nature of the sites and type of interaction. We have confirmed thirteen unique sites of inducer-sensitive protection along the operator sequence (+1, 2, 3, 4, 6, 8, 13, 15, 16, 18, 19, 20, 21) using this method compared to per-substitution with BrdU (Ogata and Gilbert, 1977). The ability of these photosensitive DNAs to form short-range cross-links to bound protein has been used to determine the efficiency with which cross-linked protein-DNA complexes are generated at each individual site of BrdU substitution. Five sites of high efficiency cross-linking to the repressor protein have been identified (+3, 4, 14, 18, 19). Comparison of the UV protection results and the cross-linking data shows that these processes provide complementary tools for identifying and analyzing individual protein-DNA contacts.
Using these same BrdU-substituted operator DNAs, we attempted to define individual protein-DNA interactions with respect to the specific amino acid(s) making contact at a selected site within the operator sequence. With the selection of the T$\sb{+3}$ site for our initial investigation, the cross-linked complex was formed and isolated. These polypeptide-DNA species were prepared for final analysis through a series of steps including proteolysis and anion-exchange HPLC. Protein sequence analysis on the purified peptide-operator complex identified a peptide spanning Val23 through Lys33. The data suggest His29 as the specifically crosslinked amino acid.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/16407 |
| Date | January 1990 |
| Creators | Wick, Kyle Lynn |
| Contributors | Matthews, Kathleen S. |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | 177 p., application/pdf |
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