title: Structural basis for RNA-genome recognition during bacteriophage Qβ replication.
abstract: Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qβ replicase holoenzyme complex, which is responsible for amplifying the Qβ (+)-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 β-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1-2) in the recognition of Qβ (+)-RNA by the Qβ replicase complex. We show how three basic residues of the β 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 Qβ 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 β-subunit and protein S1 cooperatively bind the (+)-stranded Qβ genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Heidi
authors.initials: H
authors.lastname: Gytz
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Durita
authors.initials: D
authors.lastname: Mohr
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Paulina
authors.initials: P
authors.lastname: Seweryn
authors.affiliation: Interdisciplinary Nanoscience Centre (iNANO) and Department of Chemistry, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Yoshimura
authors.affiliation: Institute of Protein Research of the Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.
authors.forename: Zarina
authors.initials: Z
authors.lastname: Kutlubaeva
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Fleur
authors.initials: F
authors.lastname: Dolman
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Bosene
authors.initials: B
authors.lastname: Chelchessa
authors.affiliation: Institute of Protein Research of the Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Chetverin
authors.affiliation: Interdisciplinary Nanoscience Centre (iNANO) and Department of Chemistry, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Frans A A
authors.initials: FA
authors.lastname: Mulder
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Ditlev E
authors.initials: DE
authors.lastname: Brodersen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark crk@mbg.au.dk.
authors.forename: Charlotte R
authors.initials: CR
authors.lastname: Knudsen
mesh_terms: D017908:Allolevivirus / Q000235:genetics / Q000502:physiology*; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D016679:Genome, Viral; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011777:Q beta Replicase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism; D014779:Virus Replication
chemical_list: D029968:Escherichia coli Proteins; D021122:Protein Subunits; D012367:RNA, Viral; D012269:Ribosomal Proteins; C544829:ribosomal protein S1, E coli; D011777:Q beta Replicase