The rate of dissociation of P1, the 5′ product of hammerhead cleavage, is 100?300-fold slower in full-length hammerheads than in hammerheads that either lack or have disrupting mutations in the loop?loop tertiary interaction. The added stability requires the presence of residue 17 at the 3′ terminus of P1 but not the 2′, 3′ terminal phosphate. Since residue 17 is buried within the catalytic core of the hammerhead in the X-ray structure, we propose that the enhanced P1 stability is a result of the cooperative folding of the hammerhead around this residue. However, since P1 is fully stabilized at >2.5 mM MgCl2 while hammerhead activity continues to increase with an increase in MgCl2 concentration, it is clear that the hammerhead structure in the transition state must differ from that of the product complex. The product stabilization assay is used to test our earlier proposal that different tertiary interactions modulate the cleavage rate by differentially stabilizing the core.
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