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The binding interaction of HMG-1 with the TATA-binding protein/TATA complex
1Department of Chemistry, Bowling Green State University, Bowling Green, Ohio 43403, USA.
The Journal of Biological Chemistry
|June 8, 2001
Summary
High mobility group box 1 (HMG-1) protein represses transcription by binding to TATA-binding protein (TBP). This interaction, primarily involving HMG-1's acidic C-terminus and TBP's N-terminus (Q-tract), enhances TBP binding affinity to DNA.
Area of Science:
- Molecular Biology
- Gene Regulation
- Protein-DNA Interactions
Background:
- High mobility group box 1 (HMG-1) protein is known to regulate transcription mediated by RNA polymerase II.
- HMG-1 can function as a transcriptional repressor by forming complexes with other proteins.
- The TATA-binding protein (TBP) is a crucial component of the transcription preinitiation complex.
Purpose of the Study:
- To identify the specific domains of HMG-1 and TBP involved in the formation of the HMG-1/TBP/TATA complex.
- To elucidate the functional consequences of HMG-1 binding on TBP's affinity for the TATA element.
- To map the precise binding site of HMG-1 on TBP and assess the evolutionary conservation of this interaction.
Main Methods:
- Electrophoretic mobility shift assays (EMSA) were employed to study protein-DNA complex formation.
- Site-directed mutagenesis and deletion analysis were used to map protein interaction domains.
- Competition assays with protein fragments and antibodies were performed.
Main Results:
- The acidic C-terminal domain of HMG-1 and the N-terminus of human TBP are essential for stable HMG-1/TBP/TATA complex formation.
- HMG-1 binding increases TBP's affinity for the TATA element approximately 20-fold, primarily by accelerating the binding rate.
- HMG-1 specifically binds to the Q-tract region (amino acids 55-95) of human TBP, and this binding is competed by a specific monoclonal antibody (1C2).
- Stable complex formation occurs with TBP from human and Drosophila but not yeast, highlighting the Q-tract's importance and evolutionary conservation.
Conclusions:
- The N-terminal Q-tract of TBP is the primary binding site for HMG-1 during transcriptional repression.
- HMG-1 acts as a potent modulator of TBP-TATA element interactions, influencing transcription initiation.
- The HMG-1/TBP interaction is conserved across species, with notable exceptions like yeast, underscoring the functional significance of the Q-tract.