ABSTRACT
Upon infection of Escherichia coli by bacteriophage Qß, the virus-encoded ß-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qß replicase holoenzyme complex, which is responsible for amplifying the Qß (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the ß-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1-2) in the recognition of Qß (+)-RNA by the Qß replicase complex. We show how three basic residues of the ß 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 Qß 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 ß-subunit and protein S1 cooperatively bind the (+)-stranded Qß genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.
Subject(s)
Allolevivirus/physiology , Genome, Viral , Q beta Replicase/chemistry , Virus Replication , Allolevivirus/genetics , Dimerization , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Mutation , Protein Binding , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Q beta Replicase/genetics , Q beta Replicase/metabolism , RNA, Viral/biosynthesis , RNA, Viral/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolismABSTRACT
S1 is the largest ribosomal protein, and is vitally important for the cell. S1 is also a subunit of Qß replicase, the RNA-directed RNA polymerase of bacteriophage Qß. In both protein and RNA syntheses, S1 is commonly believed to bind to a template RNA at the initiation step, and not to be involved in later events. Here, we show that in Qß replicase-mediated RNA synthesis, S1 functions at the termination step by promoting release of the product strand in a single-stranded form. This function is fulfilled by the N-terminal fragment comprising the first two S1 domains. The results suggest that S1 might also have a role other than mRNA binding in the ribosome.
