Base-pairing potential identified by in vitro selection predicts the kinked RNA backbone observed in the crystal structure of the alfalfa mosaic virus RNA-coat protein complex.
The three-dimensional structure of the 3' terminus of alfalfa mosaic virus RNA in complex with an amino-terminal coat protein peptide revealed an unusual RNA fold with inter-AUGC basepairing stabilized by key arginine residues (Guogas, et al., 2004). To probe viral RNA interactions with the full-length coat protein, we have used in vitro genetic selection to characterize potential folding patterns among RNAs isolated from a complex randomized pool. Nitrocellulose filter retention, electrophoretic mobility bandshift analysis, and hydroxyl radical footprinting techniques were used to define binding affinities and to localize the potential RNA-protein interaction sites. Minimized binding sites were identified that included both the randomized domain and a portion of the constant regions of the selected RNAs. The selected RNAs, identified by their ability to bind full-length coat protein, have the potential to form the same unusual inter-AUGC Watson-Crick base pairs observed in the crystal structure, although the primary sequences diverge from the wild-type RNA. A constant feature of both the wild-type RNA and the selected RNAs is a G ribonucleotide in the third position of an AUGC-like repeat. Competitive binding assays showed that substituting adenosine for the constant guanosine in either the wild-type or selected RNAs impaired coat protein binding. These data suggest that the interactions observed in the RNA-peptide structure are likely recapitulated when the full-length protein binds. Further, the results underscore the power of in vitro genetic selection for probing RNA-protein structure and function.
Boyce
Michael
M
Harvard-MIT Division of Health Sciences and Technology, and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02139, USA.
Scott
Felicia
F
Guogas
Laura M
LM
Gehrke
Lee
L
D017794:Alfalfa mosaic virus / Q000235:genetics* / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D018983:DNA Footprinting; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D052156:SELEX Aptamer Technique
D036022:Capsid Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C095691:coat protein, Alfalfa mosaic virus