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4‐thio‐U cross‐linking identifies the active site of the VS ribozyme

To identify nucleotides in or near the active site, we have used a circularly permuted version of the VS ribozyme capable of cleavage and ligation to incorporate a single photoactive nucleotide analog, 4‐thio‐ uridine, immediately downstream of the scissile bond. Exposure to UV light produced two cross‐linked RNAs, in which the 4‐thio‐uridine was cross‐linked to A756 in the 730 loop of helix VI. The cross‐links formed only under conditions that support catalytic activity, suggesting that they reflect functionally relevant conformations of the RNA. One of the cross‐linked RNAs contains a lariat, indicative of intramolecular cross‐linking in the ligated RNA; the other is a branched molecule in which the scissile phosphodiester bond is cleaved, but occupies the same site in the ribozyme–substrate complex. These are the two forms of the RNA expected to be the ground state structures on either side of the transition state. This localization of the active site is consistent with previous mutational, biochemical and biophysical data, and provides direct evidence that the cleavage site in helix I interacts with the 730 loop in helix VI.