Robust and generic RNA modeling using inferred constraints: a structure for the hepatitis C virus IRES pseudoknot domain

Abstract

RNA function is dependent on its structure, yet three-dimensional folds for most biologically important RNAs are unknown. We develop a generic discrete molecular dynamics-based modeling system that uses long-range constraints inferred from diverse biochemical or bioinformatic analyses to create statistically significant (p < 0.01) nativelike folds for RNAs of known structure ranging from 45 to 158 nucleotides. We then predict the unknown structure of the hepatitis C virus internal ribosome entry site (IRES) pseudoknot domain. The resulting RNA model rationalizes independent solvent accessibility and cryo-electron microscopy structure information. The pseudoknot domain positions the AUG start codon near the mRNA channel and is tRNA-like, suggesting the IRES employs molecular mimicry as a functional strategy.

Figure 1

Generic constraint system. (A) Three-bead model for RNA. (B) Interaction potential for distance constraints.

Figure 2

Comparison of predicted and experimental RNA structures. Spheres indicate phosphate groups. RMSDs for the CrPV, HHR, tRNA and P546 RNAs are 3.6, 5.4, 6.4, and 11.3 Å.

Figure 3

RNA structure prediction for the pseudoknot domain of the HCV IRES. (A) Secondary structure (10). (B) Predicted structure of the uncomplexed pseudoknot domain. Spheres indicate phosphate positions; nucleotides are colored as shown in panel A. (C) HCV-PK model placed into the electron density of the IRES-ribosome complex (12b). (D) Superposition of hydroxyl radical protection data (11) on the HCV-PK RNA model. Spheres correspond to protected sugar pseudo-atoms; red and yellow indicate strong and moderate protection, respectively.

Figure 4

Docking of HCV IRES RNA into the mRNA channel of the 40S ribosome. Cartoons of the 40S subunit (black) and HCV IRES (gray) are based on cryo-EM studies (12). The AUG start site codon, mRNA entry and exit sites, and tRNA binding sites are labeled on the 40S subunit. The HCV-PK model is colored and positioned in the same orientation as in Figure 3C.