The cocrystal structure of the PP7 bacteriophage coat protein in complex with its translational operator identifies a distinct mode of sequence-specific RNA recognition when compared to the well-characterized MS2 coat protein-RNA complex. The structure reveals the molecular basis of the PP7 coat protein's ability to selectively bind its cognate RNA, and it demonstrates that the conserved β-sheet surface is a flexible architecture that can evolve to recognize diverse RNA hairpins.The inherent sequence diversity generated by RNA replication, which allows RNA viruses to evolve, must be tempered by the need for conservation of functional elements within the viral genome. The coat proteins of single-stranded (ss) RNA bacteriophages have both structural and regulatory roles in the viral life cycle, as they assemble into the mature capsid and regulate translation of the phage replicase through binding and sequestration of an RNA hairpin that contains the initiation site 1 . The coat protein and translational operator form a pair that must coevolve, so that mutation in one must be accompanied by compensatory mutations in the other. The Pseudomonas aeruginosa bacteriophage PP7 is a model system for exploring coevolution because of the extensive sequence divergence of both the PP7 coat protein and translational operator from other ssRNA bacteriophages 2 . The PP7 and MS2 coat proteins share only 15% sequence identity, and their cognate RNA hairpins differ in the position of the bulged adenosine as well as in the size and nucleotide composition of the loop (Fig. 1a). Biochemical experiments have shown that both coat proteins bind their own RNA hairpins with high affinity (K d ~ 1 nM) and are able to discriminately bind in favor of their own RNA by ~1,000-fold 3,4 . In contrast to the PP7 coat protein, the Qβ coat protein also shares low sequence identity (21%) with the MS2 coat protein, yet it uses an RNAbinding mode similar to that of the MS2 coat protein [5][6][7] Fig. 1 and Supplementary Methods online). PP7ΔFG was generated by removing residues 67-75 and, notably, this truncation binds its translational operator with affinity similar to that of the fulllength protein ( Supplementary Fig. 2 online). The PP7ΔFG monomer adopts the topology characteristic of ssRNA bacteriophage coat proteins, with an N-terminal β hairpin, a fivestranded antiparallel β sheet and two C-terminal α helices ( Supplementary Fig. 3a online) [8][9][10][11] . Antiparallel association of protomers in the dimer positions the last β strands adjacent to one another, resulting in a ten-stranded β sheet that comprises the RNA-binding surface. The interwoven packing of the α helices between each other and the N-terminal β hairpins further stabilizes the dimer, combining with the β sheet interface to bury ~3,000 Å 2 of solvent-accessible surface area. The PP7ΔFG dimer has almost identical free and bound structures (r.m.s. deviation < 0.9 Å for all Cα atoms) and is similar to that of the MS2 coat protein (r.m.s. deviation of < 2.3 Å for all Cα atoms...