Altered 3'-terminal RNA structure in phage Qbeta adapted to host factor-less Escherichia coli.
The RNA phage Qbeta requires for the replication of its genome an RNA binding protein called Qbeta host factor or Hfq protein. Our previous results suggested that this protein mediates the access of replicase to the 3'-end of the Qbeta plus strand RNA. Here we report the results of an evolutionary experiment in which phage Qbeta was adapted to an Escherichia coli Q13 host strain with an inactivated host factor (hfq) gene. This strain initially produced phage at a titer approximately 10,000-fold lower than the wild-type strain and with minute plaque morphology, but after 12 growth cycles, phage titer and plaque size had evolved to levels near those of the wild-type host. RNAs isolated from adapted Qbeta mutants were efficient templates for replicase without host factor in vitro. Electron microscopy showed that mutant RNAs, in contrast to wild-type RNA, efficiently interacted with replicase at the 3'-end in the absence of host factor. The same set of four mutations in the 3'-terminal third of the genome was found in several independently evolved phage clones. One mutation disrupts the base pairing of the 3'-terminal CCCOH sequence, suggesting that the host factor stimulates activity of the wild-type RNA template by melting out its 3'-end.
Schuppli
D
D
Institut für Molekularbiologie, Universität Zürich, Hönggerberg, 8093 Zürich, Switzerland.
Miranda
G
G
Tsui
H C
HC
Winkler
M E
ME
Sogo
J M
JM
Weber
H
H
D001483:Base Sequence; D002352:Carrier Proteins / Q000502:physiology; D004926:Escherichia coli / Q000821:virology*; D029968:Escherichia coli Proteins; D035001:Host Factor 1 Protein; D034982:Integration Host Factors; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010641:Phenotype; D012317:RNA Phages / Q000235:genetics* / Q000648:ultrastructure; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D016601:RNA-Binding Proteins / Q000502:physiology
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D035001:Host Factor 1 Protein; D034982:Integration Host Factors; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C440502:integration host factor, E coli