Mutants of the bacteriophage MS2 coat protein that alter its cooperative binding to RNA.
An RNA binding assay measuring cooperative protein binding has been used to evaluate the effects of mutations in the MS2 phage coat protein expected to disrupt capsid assembly. By using the crystal structure of the virus as a guide, six different mutations in the FG loop structure were selected in which hydrophobic residues were replaced with charged residues. Most of these proteins form capsids in Escherichia coli, but not in an in vitro assembly assay, suggesting that interdimer interactions are weaker than wild type. These mutant proteins reduce the free energy of cooperative protein binding to a double-hairpin RNA from its wild-type value of -1.9 kcal/mol. Several of the variants that have large effects on cooperativity have no effect on RNA affinity, suggesting that protein-RNA interactions can be affected independently of dimer-dimer interactions. The V75E;A81G protein, which shows no measurable cooperativity, binds operator RNA equally well as the wild-type protein under a variety of buffer conditions. Because this protein also exhibits similar specificity for variant RNA sequences, it will be useful for studying RNA binding properties independent of capsid assembly.
LeCuyer
K A
KA
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Behlen
L S
LS
Uhlenbeck
O C
OC
D001483:Base Sequence; D002213:Capsid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D009690:Nucleic Acid Conformation; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D036022:Capsid Proteins; D046911:Macromolecular Substances; D016601:RNA-Binding Proteins; D012313:RNA