Solution structure and functional analysis of a frameshift-stimulating RNA pseudoknot from sugarcane yellow leaf virus

Plant luteoviral RNA viruses employ -1 frameshifting for the production of P1 and P1-P2 fusion proteins important for viral replication. Luteoviral pseudoknots are characterized by three adenosines in the 3' side of loop L2 known to be important for maintaining frameshifting efficiency and pseudoknot stability. A proposed P1-P2 mRNA pseudoknot from sugarcane yellow leaf virus (ScYLV) was of interest since it contained two adenosine to cytidine substitutions in L2. Functional analysis shows that the in vitro frameshifting efficiency is greater (~15%) than any other luteoviral pseudoknot. The NMR-derived solution structure of the ScYLV RNA pseudoknot shows that C25 is looped out of the triplex structure and the 3' most L2 cytidine (C27) and A24 form cis Watson-Crick/sugar-edge interactions with C14 and C15 in stem S1, respectively. Thus, the ScYLV pseudoknot maintains a similar triple helical architecture as other luteoviral pseudoknots. Surprisingly, the frameshifting efficiency of the C27A ScYLV pseudoknot is decreased by ~8 fold relative to wild-type ScYLV. The solution structure of the C27A ScYLV RNA exhibits a global fold similar to the wild-type RNA; however, distinct hydrogen bonding interactions at the helical junction are observed. Specifically, C8+ in the C8+ major groove base triple moves ~2.3? relative to the accepting (G12-C28) base pair relative to the WT RNA. New NMR experiments have been developed and/or applied to confirm Watson-Crick base pairs and tertiary structural interactions in the PEMV-1 and ScYLV pseudoknots by direct observation of trans hydrogen bond scalar couplings. In addition, intrabase couplings in cytidine and adenosine have been measured, providing a valuable tool for the assignment of amino and N3/N1 resonances in RNA. Finally, thermodynamic analysis of the pairwise coupling between the major groove and minor groove tertiary structural hydrogen bonds at the helical junction have been investigated by monitoring the thermal unfolding of WT, dC14, C27A, and dC14/C27A RNAs as a function of pH. Favorable pairwise coupling characterized the WT ScYLV and BWYV RNAs, while unfavorable coupling characterized the poorly functional C27A ScYLV RNA. The implications of these structural, functional, and thermodynamic findings on the mechanism of frameshift stimulation is discussed.