Bacteriophage T4 regA protein binds RNA as a monomer, overcoming dimer interactions

Area of Science:

• Molecular Biology
• Protein-RNA Interactions
• Biochemistry

Background:

• The T4 translational repressor protein regA regulates gene expression by binding specific RNA elements.
• Understanding the oligomeric state of regA during RNA binding is crucial for elucidating its regulatory mechanism.

Purpose of the Study:

• To determine the stoichiometry of the complex formed between regA protein and the gene 44 recognition element (gene 44RE) RNA.
• To investigate the oligomeric state of regA in solution and upon RNA binding.

Main Methods:

• Quantitative binding assays to determine protein-RNA stoichiometry.
• Gel filtration chromatography (Sephadex G-75) to assess protein and complex molecular mass.
• Covalent crosslinking with glutaraldehyde to analyze protein oligomerization in solution and upon RNA interaction.

Main Results:

• RegA protein binds gene 44RE RNA with a 1:1 stoichiometry under quantitative conditions.
• Gel filtration indicates free regA exists as a dimer (29 kDa), while the regA-RNA complex is monomeric (20 kDa).
• Covalent crosslinking confirms regA dimer formation in solution, but RNA addition inhibits dimer crosslinking, suggesting disrupted protein-protein interactions.

Conclusions:

• The translational repressor regA binds gene 44RE RNA as a monomer.
• RegA protein-protein interactions in solution are disrupted or blocked by gene 44RE RNA binding.
• These findings clarify the functional oligomeric state of regA during translational repression.