Modification of cytidines in a Q beta replicase template: analysis of conformation and localization of lethal nucleotide substitutions.

The solution conformation of MDV-1( + ) RNA, a small RNA template replicated autocatalytically in vitro by Q beta replicase, was investigated with sodium bisulfite, a reagent that selectively converts single-stranded cytidines to uridines. The reactivity of 45 of the 76 cytidines in MDV-1( + ) RNA was determined by nucleotide sequence analysis. Only 14 of these 45 cytidines were converted to uridine. Treatment of the RNA with methoxyamine, another single-strand-specific cytidine modification reagent, gave results in good agreement with the bisulfite data. The limited reactivity of MDV-1 ( + ) RNA with these reagents indicates that it is a highly structured molecule. A secondary structure consistent with the chemical modification data is proposed. Modification of MDV-1 ( + ) RNA by bisulfite renders it inactive as a template for RNA replication. This inactivation and the modification of the cytidines at the 3' end of the molecule occur at very similar rates. By using a short complementary RNA "mask" to protect just these cytidines, we demonstrated that the loss of activity resulted from their modification. This implies that one or more of the cytidines in the 3'-terminal sequence is required for template activity and that changes within this sequence can have lethal consequences. The effects of modification elsewhere in the sequence are discussed.