Structure, folding and activity of the VS ribozyme: importance of the 2-3-6 helical junction

The VS nucleolytic ribozyme has a core comprising five helices organized by two three-way junctions. The ribozyme can act in trans on a hairpin-loop s ubstrate, with which it interacts via tertiary contacts. We have determined that one of the junctions (2-3-6) undergoes two-stage ion-dependent foldin g into a stable conformation, and have determined the global structure of t he folded junction using long-range distance restraints derived from fluore scence resonance energy transfer. A number of sequence variants in the junc tion are severely impaired in ribozyme cleavage, and there is good correlat ion between changes in activity and alteration ill the folding of junction 2-3-6. These studies point to a special importance of G and A nucleotides i mmediately adjacent to helix: II, and comparison with a similar junction of known structure indicates that this could adopt a guanine-wedge structure, We propose that the 2-3-6 junction organizes important aspects of the stru cture of the ribozyme to facilitate productive association with the substra te, and suggest that this results in an interaction between the substrate a nd the A730 loop to create the active compiler.