Dataset Information

Structural basis for RNA-genome recognition during bacteriophage Q? replication.

ABSTRACT: Upon infection of Escherichia coli by bacteriophage Q?, the virus-encoded ?-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Q? replicase holoenzyme complex, which is responsible for amplifying the Q? (+)-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 ?-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.

SUBMITTER: Gytz H

PROVIDER: S-EPMC4678825 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Structural basis for RNA-genome recognition during bacteriophage Qβ replication.

Gytz Heidi H Mohr Durita D Seweryn Paulina P Yoshimura Yuichi Y Kutlubaeva Zarina Z Dolman Fleur F Chelchessa Bosene B Chetverin Alexander B AB Mulder Frans A A FA Brodersen Ditlev E DE Knudsen Charlotte R CR

Nucleic acids research 20151117 22

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-oligosacc ...[more]

PMID: 26578560

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Project description:UnlabelledChanges in protein function and other biological properties, such as RNA structure, are crucial for adaptation of organisms to novel or inhibitory environments. To investigate how mutations that do not alter amino acid sequence may be positively selected, we performed a thermal adaptation experiment using the single-stranded RNA bacteriophage Qβ in which the culture temperature was increased from 37.2°C to 41.2°C and finally to an inhibitory temperature of 43.6°C in a stepwise manner in three independent lines. Whole-genome analysis revealed 31 mutations, including 14 mutations that did not result in amino acid sequence alterations, in this thermal adaptation. Eight of the 31 mutations were observed in all three lines. Reconstruction and fitness analyses of Qβ strains containing only mutations observed in all three lines indicated that five mutations that did not result in amino acid sequence changes but increased the amplification ratio appeared in the course of adaptation to growth at 41.2°C. Moreover, these mutations provided a suitable genetic background for subsequent mutations, altering the fitness contribution from deleterious to beneficial. These results clearly showed that mutations that do not alter the amino acid sequence play important roles in adaptation of this single-stranded RNA virus to elevated temperature.ImportanceRecent studies using whole-genome analysis technology suggested the importance of mutations that do not alter the amino acid sequence for adaptation of organisms to novel environmental conditions. It is necessary to investigate how these mutations may be positively selected and to determine to what degree such mutations that do not alter amino acid sequences contribute to adaptive evolution. Here, we report the roles of these silent mutations in thermal adaptation of RNA bacteriophage Qβ based on experimental evolution during which Qβ showed adaptation to growth at an inhibitory temperature. Intriguingly, four synonymous mutations and one mutation in the untranslated region that spread widely in the Qβ population during the adaptation process at moderately high temperature provided a suitable genetic background to alter the fitness contribution of subsequent mutations from deleterious to beneficial at a higher temperature.

Dataset Information

Structural basis for RNA-genome recognition during bacteriophage Q? replication.

Publications

Structural basis for RNA-genome recognition during bacteriophage Qβ replication.

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