CRYSTAL-STRUCTURES OF AN A-FORM DUPLEX WITH SINGLE-ADENOSINE BULLIES AND A CONFORMATIONAL BASIS FOR SITE-SPECIFIC RNA SELF-CLEAVAGE

Background: Bulged nucleotides are common secondary structural motifs in RNA molecules and are often involved in RNA-RNA and RNA-protein int eractions. RNA is selectively cleaved at bulge sites (when compared to other sites within stems) in the presence of divalent metal cations. The effects of bulge nucleotides on duplex stability and topology have been extensively investigated, but no detailed X-ray structures of bu lge-containing RNA fragments have been available. Results: We have cry stallized a self-complementary RNA-DNA chimeric 11-nucleotide sequence containing single-adenosine bulges under two different conditions, gi ving two distinct crystal forms, In both lattices the adenosines are l ooped out, leaving the stacking interactions in the duplex virtually u naffected. The bulges cause the duplex to kink in both cases, In one o f the structures, the conformation of the bulged nucleotide places its modeled 2'-oxygen in line with the adjacent phosphate on the 3' side, where it is poised for nucleophilic attack. Conclusions: Single adeno sine bulges cause a marked opening of the normally narrow RNA major gr oove in both crystal structures, rendering the bases more accessible t o interacting molecules compared with an intact stem, The geometries a round the looped-out adenosines are different in the two crystal forms , indicating that bulges can confer considerable local plasticity on t he usually rigid RNA double helix. The results provide a conformationa l basis for the preferential, metal-assisted self-cleavage of RNA at b ulged sites.