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

Abstract

The solution conformation of MDV-1(+) RNA, a small RNA template replicated auto-catalytically 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. Using a short complementary RNA mask to protect just these cytidines showed that the loss of activity resulted from their modification. This implies that 1 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.