A mutant viral RNA promoter with an altered conformation retains efficient recognition by a viral RNA replicase through a solution-exposed adenine

Abstract

Brome mosaic virus (BMV) genomic minus-strand RNA synthesis requires an RNA motif named stem-loop C (SLC). An NMR-derived solution structure of SLC was reported by Kim et al. (Nature Struc Biol, 2000, 7:415–423) to contain three replicase-recognition elements, the most important of which is a stable stem with a terminal trinucleotide loop, 5′AUA3′. The 5′-most adenine of the triloop is rigidly fixed to the stem helix by interactions that require the 3′-most adenine, which is called a clamped adenine motif. However, a change of the 3′ adenine to guanine (5′AUG3′) unexpectedly directed RNA synthesis at 130% of wild type (Kim et al., Nature Struc Biol, 2000, 7:415–423). To understand how RNA with the AUG mutation maintains interaction with the BMV replicase, we used NMR and other biophysical techniques to elucidate the solution conformation of a 13-nt RNA containing the AUG triloop, called S-AUG. We found that S-AUG has a drastically different loop conformation in comparison to the wild type, as evidenced by an unusual C[bull ]G loop-closing base pair. Despite the conformational change, S-AUG maintains a solution-exposed adenine similar to the clamped adenine motif found in the wild type. Biochemical studies of the 5′AUG3′ loop with various substitutions in the context of the whole SLC construct confirm that the clamped adenine motif exists in S-AUG remains a primary structural feature required for RNA synthesis by the BMV replicase.