X-ray crystallographic observation of "in-line" and "adjacent" conformations in a bulged self-cleaving RNA/DNA hybrid

The RNA strand in an RNA/DNA duplex with unpaired ribonucleotides can under go self-cleavage at bulge sites in the presence of a variety of divalent me tal ions (Husken et al., Biochemistry, 1996, 35:16591-16600), Transesterifi cation proceeds via an in-line mechanism, with the 2 ' -OH of the bulged nu cleotide attacking the 3 ' -adjacent phosphate group. The site-specificity of the reaction is most likely a consequence of the greater local conformat ional freedom of the RNA backbone in the bulge region. A standard A-form ba ckbone geometry prohibits formation of an in-line arrangement between 2 ' - oxygen and phosphate, However, the backbone in the region of an unpaired nu cleotide appears to be conducive to an in-line approach. Therefore, the bul ge-mediated phosphoryl transfer reaction represents one of the simplest RNA self-cleavage systems. Here we focus on the conformational features of the RNA that underlie site-specific cleavage. The structures of an RNA/DNA dup lex with single ribo-adenosyl bulges were analyzed in two crystal forms, pe rmitting observation of 10 individual conformations of the RNA bulge moiety , The bulge geometries cover a range of relative arrangements between the 2 ' -oxygen of the bulged nucleotide and the P-O5 ' bond (including adjacent and near in-line) and give a detailed picture of the conformational change s necessary to line up the 2 ' -OH nucleophile and scissile bond. Although metal ions are of crucial importance in the catalysis of analogous cleavage reactions by ribozymes, it is clear that local strain or conformational fl exibility in the RNA also affect cleavage selectivity and rate (Soukup & Br eaker, RNA, 1999, 5:1308-1325), The geometries of the RNA bulges frozen out in the crystals provide snapshots along the reaction pathway prior to the transition state of the phosphoryl transfer reaction.