Multiple folded conformations of a hammerhead ribozyme domain under cleavage conditions.

The conformation of a hammerhead ribozyme domain, formed between a 35-mer ribozyme and its 14-mer substrate, was studied under cleavage conditions with non-cleavable substrate analogues. Each analogue substrate contained a single 2'-deoxy-4-thiouridine that formed specific intermolecular crosslinks within the ribozyme-substrate complex upon irradiation with 365 nm light. The residues at positions 7 and 8 to 9 (the cleavage site) were found in contact with several bases of the ribozyme 5' conserved region regardless of whether the substrate was all-RNA, with a single deoxynucleotide at the cleavage site, or all-DNA. These contacts were observed in the presence of either 20 mM Mg2+ or 200 mM Na+. The multiple crosslinks generated between the ribozyme central core and each of the three substrates suggest the existence of several folded conformers of the ribozyme. A ribozyme mutation (A28U), which abolishes the catalytic activity, was shown to strongly affect the pattern of crosslinks; this argues for the presence of multiple folded conformers of the ribozyme some of which may be catalytically inactive.