Structural characterization of an intermolecular RNA-RNA interaction involved in the transcription regulation element of a bipartite plant virus

The 34-nucleotide trans-activator (TA) located within the RNA-2 of Red clover necrotic mosaic virus folds into a simple hairpin. The eight-nucleotide TA loop base pairs with eight complementary nucleotides in the TA binding sequence (TABS) of the capsid protein subgenomic promoter on RNA-1 and trans-activates subgenomic RNA synthesis. Short synthetic oligoribonucleotide mimics of the RNA-1 TABS and the RNA-2 TA form a weak 1:1 bimolecular complex in vitro with a K-a of 5.3 x 10(4) M-1. K-a determination for a series of RNA-1 and RNA-2 mimic variants indicated optimum stability is obtained with seven-base complementarity. Thermal denaturation and NMR show that the RNA-1 TABS 8mers are weakly ordered in solution while RNA-2 TA oligomers form the predicted hairpin. NMR diffusion studies confirmed RNA-1 and RNA-2 oligomer complex formation in vitro. MC-Sym generated structural models suggest that the bimolecular complex is composed of two stacked helices, one being the stem of the RNA-2 TA hairpin and the other formed by the intermolecular base pairing between RNA-1 and RNA-2. The RCNMV TA structural model is similar to those for the Simian retrovirus frameshifting element and the Human immunodeficiency virus-1 dimerization kissing hairpins, suggesting a conservation of form and function.