Regional mutagenesis of the gene encoding the phage Mu late gene activator C identifies two separate regions important for DNA binding

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• Bacteriophage Mu's lytic development involves a transcriptional regulatory cascade.
• Late transcription necessitates host RNA polymerase and the Mu-encoded activator protein C.
• Late promoters (P(lys), P(I), P(P), P(mom)) possess -10 hexamers but lack -35 hexamers.

Purpose of the Study:

• To investigate the DNA-binding regions of bacteriophage Mu's activator protein C.
• To test the hypothesis that helix-turn-helix and beta-strand regions of protein C are crucial for DNA binding.
• To identify specific amino acid residues involved in protein C's interaction with DNA.

Main Methods:

• Mutagenesis of the C gene using degenerate oligonucleotide-directed PCR.
• Screening of mutants for activation of a P(lys)-galK fusion.
• Analysis of mutant proteins via gel mobility shift, beta-galactosidase, and PAGE assays.

Main Results:

• Identified specific amino acid residues in both predicted DNA-binding regions of protein C.
• Demonstrated the importance of these residues for protein C's DNA-binding activity.
• Confirmed the role of helix-turn-helix and beta-strand regions in protein C's function.

Conclusions:

• Specific regions and amino acid residues within bacteriophage Mu's protein C are critical for DNA binding.
• This study elucidates the molecular mechanism of late gene activation by protein C.
• Findings contribute to understanding bacteriophage regulatory systems and DNA-protein interactions.