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, , and 21 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 N-and P22 N-deficient viruses, but no proteins were found to complement both P22 N-and 21 N-deficient viruses. A single tryptophan substitution allowed P22 N to complement both P22 and N ؊ . The existence of relaxed-specificity mutants suggests that conformational plasticity provides evolutionary transitions between distinct modes of RNA-protein recognition.Lambdoid phages , P22, 21, and other phages regulate the expression of delayed early genes by allowing transcription past terminators in the P left and P right operons. These phages share regulatory mechanisms with , but they have uncertain evolutionary relationships that are obscured by recombination among tailed phages (Caudovirales) (7,8). In phage , antitermination allows expression of genes regulating the development of lysis or lysogeny (12). The assembly of transcription antitermination complexes in P22, , and 21 is initiated by the binding of viral N proteins to small hairpin boxB RNAs in the nut sites (N utilization) of regulated transcripts (40). These complexes contain N and host factors, including NusA and the transcribing polymerase, allowing transcription to proceed through downstream transcription termination signals. P22, , and 21 exhibit type specificity, where the N protein of one virus cannot complement its absence in a different virus (13,24). N proteins recognize their cognate boxB RNAs via argininerich domains near their amino termini (21). Their boxBs are hairpin RNAs that have little sequence similarity, yet similar secondary structures (Fig. 1A). Alignment of P22, , and 21 reveals that N protein RNA-binding domains contain four conserved amino acids (Fig. 1B). Protein and RNA sequence differences create specificity; noncognate interactions functi...