title: Asymmetric contributions to RNA binding by the Thr(45) residues of the MS2 coat protein dimer.
abstract: A prominent feature of the interaction of MS2 coat protein with RNA is the quasi-symmetric insertion of a bulged adenine (A-10) and a loop adenine (A-4) into conserved pockets on each subunit of the coat protein dimer. Because of its presence in both of these adenine-binding pockets, Thr(45) is thought to play an important role in interaction with RNA on both subunits of the dimer. To test the significance of Thr(45), we introduced all 19 amino acid substitutions. However, we were initially unable to determine the effects of the mutations on RNA binding because every substitution compromised the ability of coat protein to fold correctly. Genetic fusion of coat protein subunits reverted these protein structural defects, allowing us to show that the RNA binding activity of coat protein tolerates substitution of Thr(45), but only on one or the other subunit of the dimer. Single-chain heterodimer complementation experiments suggest that the primary site of Thr(45) interaction with RNA is with A-4 in the translational operator. Either contact of Thr(45) with A-10 makes little contribution to stability of the RNA-protein complex, or the effects of Thr(45) substitution are offset by conformational adjustments that introduce new, favorable contacts at nearby sites.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA. dpeabody@salud.unm.edu
authors.forename: D S
authors.initials: DS
authors.lastname: Peabody
authors.forename: A
authors.initials: A
authors.lastname: Chakerian
mesh_terms: D002213:Capsid / Q000737:chemistry* / Q000378:metabolism*; D036022:Capsid Proteins; D019281:Dimerization; D017909:Levivirus / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D013912:Threonine
chemical_list: D036022:Capsid Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D013912:Threonine