Abstract
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.
Highlights
From the Departments of Cell Biology Albuquerque, New Mexico 87131 and Biochemistry, University of New Mexico School of Medicine, The coat protein of bacteriophage
The rationale of their design was to create a situation in which the synthesis of a replicase-P-galactosidase fusion protein encoded by one plasmid is under translational control of coat protein encoded by a second plasmid
These results show that the two-plasmid expression system mimics the translational repression observed in MSB-infected cells
Summary
From the Departments of Cell Biology Albuquerque, New Mexico 87131 and Biochemistry, University of New Mexico School of Medicine, The coat protein of bacteriophage. 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. At late stages of infection of Escherichia coli by bacteriophage MS2, the viral coat protein binds a stem-loop structure in the viral RNA which contains the ribosome binding site of the replicase gene, repressing replicase synthesis [1]. The translational operator consists of a 21-nucleotide stem-loop whose specific three-dimensional structure forms the coat protein-binding site [2,3]. This small piece of RNA may serve as the signal for encapsidation of the RNA genome.
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