Protonatable Hairpins Are Conserved in the 5′-Untranslated Region of Tymovirus RNAs

The secondary structures of the 5′-untranslated region (5′-UTR) of five different tymoviruses have been determined by structure probing, computer prediction and sequence comparison. Despite large sequence differences, there are remarkable similarities in the secondary structure. In all viruses two or four hairpins are found, most of which contain a symmetrical internal loop consisting of adjacent C-C or C-A mismatches. Since it is known that such mismatches can be protonated and protonated cytosines play an important role in RNA-protein interactions in tymoviral virions, the influence of pH on the conformation of the internal loop was studied. UV melting experiments and 1-dimensional proton NMR at varying pH values and salt concentrations confirm that the hairpins can be protonated under relatively mild conditions. The hairpin found in the 5-UTR of erysimum latent virus, which has an asymmetrical internal loop consisting of cytosines and uridines, shows comparable behaviour. It is concluded that all tymoviral RNAs contain protonatable hairpins in the 5′-UTR. Binding experiments with empty viral capsids, however, do not yet establish a role in capsid protein binding.