Translational repression by bacteriophage MS2 coat protein expressed from a plasmid. A system for genetic analysis of a protein-RNA interaction.
The coat protein of bacteriophage MS2 is a translational repressor. It inhibits the synthesis of the viral replicase by binding a specific RNA structure that contains the replicase translation initiation region. In order to begin a genetic dissection of the repressor activity of coat protein, a two-plasmid system has been constructed that expresses coat protein and a replicase-beta-galactosidase fusion protein from different, compatible plasmids containing different antibiotic-resistant determinants. The coat protein expressed from the first plasmid (pCT1) represses synthesis of a replicase-beta-galactosidase fusion protein encoded on the other plasmid (pRZ5). Mutations in the translational operator or in coat protein result in constitutive synthesis of the enzyme. This permits the straightforward isolation of mutations in the coat sequence that affect repressor function. Because of the potential importance of cysteine residues for RNA binding, mutations were constructed that substitute serines for the cysteine residues normally present at positions 46 and 101. Both of these mutations result in translational repressor defects. Chromatographic and electron microscopic analyses indicate that the plasmid-encoded wild-type coat protein forms capsids in vivo. The ability of the mutants to adopt and/or maintain the appropriate conformation was assayed by comparing them to the wild-type protein for their ability to form capsids. Both mutants exhibited evidence of improper folding and/or instability as indicated by their aberrant elution behavior on a column of Sepharose CL-4B. Methods were developed for the rapid purification of plasmid-encoded coat protein, facilitating future biochemical analyses of mutant coat proteins.
Peabody
D S
DS
Department of Cell Biology, University of New Mexico School of Medicine, Albuquerque 87131.
D001435:Bacteriophages; D001483:Base Sequence; D015153:Blotting, Western; D002213:Capsid / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D014176:Protein Biosynthesis / Q000187:drug effects*; D012367:RNA, Viral / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D001616:beta-Galactosidase