The role of essential pyrimidines in the hairpin ribozyme-catalysed reaction

The hairpin ribozyme is an example of a small catalytic RNA that catalyses the endonucleolytic transesterification of RNA in a highly sequence-specific manner. We have utilised chemical synthesis of RNA to create mutants of the hairpin ribozyme in which a nucleoside analogue replaces one of the essential pyrimidines in the ribozyme. Individual pyrimidine nucleosides were substituted by 4-thiouridine, O-4-methyluridine, O-2-methyluridine or 2-pyrimidinone-1-beta-D-riboside. To facilitate the synthesis of oligoribonucleotides containing 4-thiouridine, we have devised a new synthetic route to the key intermediate 5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-S-cyanoethyl-4-thiouridine. The ability of the modified ribozymes to support catalysis was studied and the steady-state kinetic parameters were determined for each mutant. The range of analogues used in this study allows the important functional groups of the essential pyrimidines to be identified. The results demonstrate that each pyrimidine (U-41, U-42 and C-25) plays an important role in hairpin ribozyme catalysis. The findings are discussed in terms of the various models that have been proposed for loop B of the hairpin ribozyme. (C) 1999 Academic Press.