A Y form of hammerhead ribozyme trapped by photo-cross-links retains full cleavage activity.

The conformation in solution of a small bipartite I-III hammerhead ribozyme has been deduced from the photo-crosslinks formed between cleavable ribo-deoxysubstrates appropriately substituted with the probe deoxy-4-thiouridine and ribozyme residues. The ribozyme-substrate complex is able to adopt a Y-like structure with stems I and II in close proximity in the presence of 400 mM Na+ only. Indeed, a cross-link joining stem I (1.6) to loop II (AL2.4) forms in significant amount under these conditions. This cross-linked complex furthermore elicits, upon Mg2+ addition, a catalytic activity similar to that exhibited by the complexes cross-linked at the distal ends of either stem I or stem III or of the non-substituted bipartite complex. This shows that the reaction mechanism is fully compatible with a strong structural constraint between stems I and II and that sodium ions at high concentration (400 mM) are able to promote a proper folding of hammerhead ribozymes. None of the multiple cross-links formed within the ribozyme core (probe in position 16.1 or 1.1) was found catalytically active. The cross-link patterns nevertheless indicate a higher flexibility of the core in Na+ than in Mg2+. While most of the cross-links can be accommodated by the Y solution structure, some of them (16.1 to U4 and 2.1) definitely can not, suggesting that additional alternative inactive conformations exist in solution.