The evolutionary pathway from a biologically inactive polypeptide sequence to a folded, active structural mimic of...

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Bacteriophage T7 protein Ocr mimics DNA to inhibit Type I restriction/modification (RM) enzymes.
• Ocr's structure and function are linked to its acidic residues, which contribute to its DNA-mimicking electrostatic properties.

Purpose of the Study:

• To investigate the role of electrostatic interactions, specifically acidic residues, in Ocr's DNA mimicry and antirestriction activity.
• To delineate the importance of individual acidic residues for Ocr function using directed evolution.

Main Methods:

• Directed evolution of a synthetic Ocr variant (pocr) with neutral amino acids replacing acidic residues.
• In vivo selection assays to detect antirestriction activity after reintroducing acidic residues.
• Analysis of host growth to assess deleterious effects.

Main Results:

• A synthetic Ocr variant (pocr) lacking acidic residues showed no antirestriction activity.
• Directed evolution successfully restored antirestriction activity by reintroducing specific acidic residues.
• The importance of acidic residues varied depending on their location within the Ocr structure, correlating with different parts of the DNA target.

Conclusions:

• Acidic residues are essential for Ocr's DNA mimicry and its ability to inhibit Type I RM enzymes.
• The specific contribution of acidic residues is region-dependent, highlighting the intricate relationship between Ocr structure and DNA recognition.
• Understanding these residues is critical for both Ocr function and avoiding negative impacts on host cell viability.