The RNA-binding domain of bacteriophage P22 N protein is highly mutable, and a single mutation relaxes specificity toward lambda.

Antitermination in bacteriophage P22, a lambdoid phage, uses the arginine-rich domain of the N protein to recognize boxB RNAs in the nut site of two regulated transcripts. Using an antitermination reporter system, we screened libraries in which each nonconserved residue in the RNA-binding domain of P22 N was randomized. Mutants were assayed for the ability to complement N-deficient virus and for antitermination with P22 boxB(left) and boxB(right) reporters. Single amino acid substitutions complementing P22 N(-) virus were found at 12 of the 13 positions examined. We found evidence for defined structural roles for seven nonconserved residues, which was generally compatible with the nuclear magnetic resonance model. Interestingly, a histidine can be replaced by any other aromatic residue, although no planar partner is obvious. Few single substitutions showed bias between boxB(left) and boxB(right), suggesting that the two RNAs impose similar constraints on genetic drift. A separate library comprising only hybrids of the RNA-binding domains of P22, lambda, and phi21 N proteins produced mutants that displayed bias. P22 N(-) plaque size plotted against boxB(left) and boxB(right) reporter activities suggests that lytic viral fitness depends on balanced antitermination. A few N proteins were able to complement both lambda N- and P22 N-deficient viruses, but no proteins were found to complement both P22 N- and phi21 N-deficient viruses. A single tryptophan substitution allowed P22 N to complement both P22 and lambda N(-). The existence of relaxed-specificity mutants suggests that conformational plasticity provides evolutionary transitions between distinct modes of RNA-protein recognition.