Mutational analysis of a satellite phage activator

Area of Science:

• Molecular Biology
• Virology
• Protein-Nucleic Acid Interactions

Background:

• Bacteriophage P4 Delta and phiR73 Delta proteins are late gene activators essential for viral replication.
• These activators interact with the host's RNA polymerase to initiate transcription of late genes.
• Understanding these interactions is key to deciphering viral gene regulation strategies.

Purpose of the Study:

• To investigate the functional domains of the phiR73 Delta protein involved in RNA polymerase interaction and activation.
• To identify specific amino acid residues critical for Delta's activity and zinc binding.
• To compare the functional properties of phiR73 Delta with P4 Delta.

Main Methods:

• Site-directed mutagenesis of the phiR73 Delta gene.
• Phage growth assays in the presence of host RNA polymerase mutations.
• Analysis of Delta protein binding to late gene promoters.
• Spectroscopic methods to assess zinc binding to mutant Delta proteins.

Main Results:

• A V19M mutation in phiR73 Delta confers resistance to the rpoA155 host mutation, suggesting residue 19 interacts with RNA polymerase.
• Mutations at residues 19, 42, and 44 reduce late gene activation while maintaining promoter binding.
• Mutation of conserved cysteine residues essential for zinc binding abolishes Delta activity and zinc coordination.

Conclusions:

• Specific regions of phiR73 Delta, including residue 19, are critical for RNA polymerase interaction and transcriptional activation.
• The conserved cysteine residues are essential for zinc binding, which is required for Delta activator function.
• These findings provide insights into the mechanism of Delta-mediated transcriptional activation in bacteriophages.