Structural basis for RNA-genome recognition during bacteriophage Q replication

Upon infection of Escherichia coli by bacteriophage Q, the virus-encoded -subunit recruits host trans-lation elongation factors EF-Tu and EF-Ts and riboso-mal protein S1 to form the Q replicase holoenzyme complex, which is responsible for amplifying the Q (+)-RNA genome. Here, we use X-ray crystallogra-phy, NMR spectroscopy, as well as sequence con-servation, surface electrostatic potential and muta-tional analyses to decipher the roles of the -subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1–2) in the recognition of Q (+)-RNA by the Q replicase complex. We show how three basic residues of the subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Q replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the -subunit and protein S1 cooperatively bind the (+)-stranded Q genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation