Complementation of RNA binding site mutations in MS2 coat protein heterodimers.
The coat protein of bacteriophage MS2 functions as a symmetric dimer to bind an asymmetric RNA hairpin. This implies the existence of two equivalent RNA binding sites related to one another by a 2-fold symmetry axis. In this view the symmetric binding site defined by mutations conferring the repressor-defective phenotype is a composite picture of these two asymmetric sites. In order to determine whether the RNA ligand interacts with amino acid residues on both subunits of the dimer and in the hope of constructing a functional map of the RNA binding site, we performed heterodimer complementation experiments. Taking advantage of the physical proximity of their N- and C-termini, the two subunits of the dimer were genetically fused, producing a duplicated coat protein which folds normally and allows the construction of the functional equivalent of obligatory heterodimers containing all possible pairwise combinations of the repressor-defective mutations. The restoration of repressor function in certain heterodimers shows that a single RNA molecule interacts with both subunits of the dimer and allows the construction of a functional map of the binding site.
Peabody
D S
DS
Department of Cell Biology, University of New Mexico School of Medicine and Cancer Research and Treatment Center, Albuquerque, NM 87131, USA.
Lim
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D003001:Cloning, Molecular; D004279:DNA, Viral; D005816:Genetic Complementation Test; D017909:Levivirus / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D011485:Protein Binding; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins; D017384:Sequence Deletion; D019065:Virus Assembly
D036022:Capsid Proteins; D004279:DNA, Viral; D016601:RNA-Binding Proteins; D012313:RNA