STRUCTURAL VARIATION INDUCED BY DIFFERENT NUCLEOTIDES AT THE CLEAVAGESITE OF THE HAMMERHEAD RIBOZYME

The hammerhead ribozyme is capable of cleaving RNA substrates at 5' UX 3' sequences (where the cleavage site, X, can be A, C, or U). Hammerh ead complexes containing dC, dA, dI, or rG nucleotides at the cleavage site have been studied by NMR. The rG at the cleavage site forms a Wa tson-Crick base pair with C3 in the conserved core of the hammerhead, indicating that rG substrates inhibit the cleavage reaction by stabili zing an inactive conformation of the molecule. isotope-edited NMR expe riments on the hammerhead complexes show that there are different shor t proton-proton distances between neighboring residues depending upon whether there is a dC or dA at the cleavage site. These NMR data demon strate that there are significant differences in the structure and/or dynamics of the active-site residues in these hammerhead complexes, Mo lecular dynamics calculations were used to model the conformations of the cleavage-site variants consistent with the NMR data. The solution conformations of the hammerhead ribozyme-substrate complexes are compa red with the X-ray structure of the hammerhead ribozyme and are used t o help understand the thermodynamic and kinetic differences among the cleavage-site variants.