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Trp repressor interaction with bromodeoxyuridine-substituted operators alters UV-induced perturbation pattern in a
1Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251.
Biochemistry
|October 12, 1993
Summary
Bromodeoxyuridine (BrdU) incorporation revealed sequence-dependent DNA structural changes upon trp repressor binding. These alterations influence repressor-operator interactions and UV photolysis patterns, indicating a complex regulatory mechanism.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- The trp repressor regulates gene expression by binding to specific DNA operator sequences.
- Understanding the precise molecular interactions between repressors and operators is crucial for deciphering gene regulation mechanisms.
- Previous studies have utilized UV crosslinking and footprinting to investigate protein-DNA interactions.
Purpose of the Study:
- To investigate DNA sites influenced by the trp repressor-operator interaction.
- To explore the structural consequences of trp repressor binding on operator DNA.
- To determine the role of DNA sequence and structure in repressor recognition.
Main Methods:
- Chemical incorporation of bromodeoxyuridine (BrdU) into trp operator DNA sequences (TrpEDCBA, TrpR, aroH).
- UV irradiation of repressor-operator complexes to induce crosslinking and strand scission.
- Analysis of protection patterns from strand scission and UV photolysis to map protein-DNA contacts and structural changes.
Main Results:
- Bromodeoxyuridine incorporation revealed differential repressor protection patterns, suggesting varied local DNA environments within operator sequences.
- UV irradiation did not result in repressor-operator crosslinking, indicating a lack of close contacts between repressor residues and thymidine 5-methyl groups in the major groove.
- UV footprinting identified 'band migration' phenomena at specific sites (e.g., CTAG sequences) upon repressor binding and UV irradiation, attributed to DNA distortion and altered backbone accessibility.
Conclusions:
- Trp repressor binding induces sequence-dependent structural alterations in operator DNA.
- These DNA structural changes, rather than direct major groove contacts, likely mediate repressor recognition and influence UV photolysis patterns.
- The findings highlight the importance of DNA conformation in protein-DNA interactions and gene regulation.