Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• Bacteriophage P22 repressor protein functions as a homodimer for DNA binding.
• Dimerization is mediated by interactions within the carboxyl (C)-terminal domain.
• Understanding these interactions is crucial for phage-P22 regulatory mechanisms.

Purpose of the Study:

• To investigate the role of the P22 repressor's C-terminal domain in dimerization and DNA binding.
• To identify amino acid regions responsible for dimerization specificity between P22 and 434 repressors.
• To explore the relationship between dimerization, cooperativity, and DNA binding site discrimination.

Main Methods:

• Construction of a plasmid (p22CT-1) for overproducing the P22 repressor's C-terminal domain (P22CT-1).
• In vitro experiments involving addition of P22CT-1 to DNA-bound P22 repressor.
• Cross-linking experiments to analyze protein-protein interactions.
• Creation and study of chimeric proteins between P22 and 434 repressors.

Main Results:

• P22CT-1 addition dissociates DNA-bound P22 repressor by forming inactive heterodimers.
• Dimerization specificity involves three distinct amino acid segments within the C-terminal domain.
• The dimerization interface may be distinct from the cooperativity interface in P22 and 434 repressors.
• Altering the dimer interface in 434 repressor impairs discrimination between its DNA binding sites.

Conclusions:

• The C-terminal domain of P22 repressor is essential for dimerization and DNA binding inhibition.
• Specific amino acid regions dictate dimerization specificity between related phage repressors.
• The C-terminal domain plays a critical role in recognizing sequence-dependent DNA structures and flexibility.