Bacteriophage P22 antitermination boxB sequence requirements are complex and overlap with those of lambda

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Transcription antitermination in bacteriophages is crucial for viral gene expression.
• Phages lambda and P22 utilize N proteins to bind boxB RNA hairpins, but the P22 interaction is less understood.
• Previous studies lacked a clear consensus sequence for P22 boxB.

Purpose of the Study:

• To elucidate the sequence requirements for the P22 N protein-boxB RNA interaction.
• To compare the binding specificities of P22 and lambda N proteins for boxB elements.
• To investigate the evolutionary flexibility of boxB sequences.

Main Methods:

• Utilized a plasmid-based antitermination system for screening boxB variants.
• Employed random loop screening and site-directed mutagenesis of boxB sequences.
• Tested binding of P22 and lambda N proteins to various boxB mutants.

Main Results:

• P22 N protein specifically recognizes GNRA-like loops in boxB RNA.
• U:A or A:U base pairs adjacent to the loop significantly modulate P22 N binding.
• Some boxB mutants exhibited cross-reactivity with both P22 and lambda N proteins.
• Single point mutations could switch boxB specificity from P22 to lambda N.

Conclusions:

• The P22 N-boxB interaction is primarily determined by the loop structure, favoring GNRA-like motifs.
• Sequence elements flanking the loop play a role in modulating N protein binding affinity.
• BoxB sequence requirements show overlap but also distinct specificities between P22 and lambda N.
• Evolutionary transitions in boxB specificity are achievable through sequential mutations, supporting neutral evolution theories.