Interactions between lac repressor protein and bromodeoxyuridine-substituted operator DNA: Identification of a specific amino acid-nucleotide contact using UV footprinting and crosslink formation

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dc.contributor.advisorMatthews, Kathleen S.en_US
dc.creatorWick, Kyle Lynnen_US
dc.date.accessioned2009-06-04T00:30:13Zen_US
dc.date.available2009-06-04T00:30:13Zen_US
dc.date.issued1990en_US
dc.description.abstractAs 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.en_US
dc.format.extent177 p.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.callnoThesis Biochem. 1990 Wicken_US
dc.identifier.citationWick, Kyle Lynn. "Interactions between lac repressor protein and bromodeoxyuridine-substituted operator DNA: Identification of a specific amino acid-nucleotide contact using UV footprinting and crosslink formation." (1990) Diss., Rice University. <a href="https://hdl.handle.net/1911/16407">https://hdl.handle.net/1911/16407</a>.en_US
dc.identifier.digitalWickKen_US
dc.identifier.urihttps://hdl.handle.net/1911/16407en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectBiochemistryen_US
dc.titleInteractions between lac repressor protein and bromodeoxyuridine-substituted operator DNA: Identification of a specific amino acid-nucleotide contact using UV footprinting and crosslink formationen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBiochemistry and Cell Biologyen_US
thesis.degree.disciplineNatural Sciencesen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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