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
✍ Scribed by Michael Boyce; Felicia Scott; Laura M. Guogas; Lee Gehrke
- Publisher
- John Wiley and Sons
- Year
- 2005
- Tongue
- English
- Weight
- 298 KB
- Volume
- 19
- Category
- Article
- ISSN
- 0952-3499
- DOI
- 10.1002/jmr.759
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✦ Synopsis
Abstract
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. Copyright © 2005 John Wiley & Sons, Ltd.