Translational Repression and Specific RNA Binding by the Coat Protein of the Pseudomonas Phage PP7

Francis Lim,David S. Peabody,Thomas P. Downey

doi:10.1074/jbc.m102411200

Abstract

PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and a distant relative to coliphages like MS2 and Qbeta. Here we show that PP7 coat protein is a specific RNA-binding protein, capable of repressing the translation of sequences fused to the translation initiation region of PP7 replicase. Its RNA binding activity is specific since it represses the translational operator of PP7, but does not repress the operators of the MS2 or Qbeta phages. Conditions for the purification of coat protein and for the reconstitution of its RNA binding activity from disaggregated virus-like particles were established. Its dissociation constant for PP7 operator RNA in vitro was determined to be about 1 nm. Using a genetic system in which coat protein represses translation of a replicase-beta-galactosidase fusion protein, amino acid residues important for binding of PP7 RNA were identified.

Highlights

PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and a distant relative to coliphages like MS2 and Q␤
We show that PP7 coat protein is a specific RNA-binding protein, capable of repressing the translation of sequences fused to the translation initiation region of PP7 replicase
Recent x-ray structure determination of RNA phages shows that homologies evident from comparisons of coat protein amino acid sequences are reflected in their tertiary structures (1–7)

Summary

EXPERIMENTAL PROCEDURES

Plasmid Constructions—The PP7 coat sequence cloned on a plasmid was kindly provided to us by Gordon Garde. After digestion with XbaI and BamHI, the fragment was cloned between the corresponding restriction sites within the polylinker of pUC119 (11), placing the coat sequence under control of the lac promoter. We constructed pP7CTNcXb by transfer of the XbaI-BamHI fragment from pETP7CTNc to pUC119 This construction retains the fusion of coat to the T7 gene 10 ribosome binding site, but returns coat to lac promoter control in pUC119. Mutants that passed this capsid assembly test were subjected to DNA sequence analysis

RESULTS

DISCUSSION

Methods