IMPORTANCE OF SPECIFIC ADENOSINE N(7)-NITROGENS FOR EFFICIENT CLEAVAGE BY A HAMMERHEAD RIBOZYME - A MODEL FOR MAGNESIUM BINDING

Five modified hammerhead ribozyme/substrate complexes have been prepared in which individual adenosine N7-nitrogens have been excised. The modified complexes were chemically synthesized with the substitution of a single 7-deazaadenosine (c7A) base analogue for residues A11, A14, A26, A27, or A28. Two of the base analogues, c7A11 and c7A14, occur in a 19-mer ribozyme, while the remaining three residues, c7A26, c7A27, and c7A28, are present in a 24-mer substrate. Under stoichiometric conditions, four of the complexes are cleaved with relatively little change in rate when compared with that of the native complex. However, the relative rate for the c7A11 complex is some 35-fold slower than that of the native complex. Steady-state kinetic analyses indicate that the cleavage efficiencies, as measured by k(cat)/K(M), for the c7A14, c7A26, c7A27, and C7A28 complexes are reduced 18-fold, 10-fold, 34-fold, and 16-fold, respectively. These reductions in cleavage efficiency are primarily a result of lower k(cat) values. By comparison, the cleavage efficiency of the C7A11 complex is reduced more than 200-fold relative to that of the native complex, again primarily as a result of a lower k(cat) value. The results suggest that the N7-nitrogen of A11 in the hammerhead ribozyme/substrate complex is critical for efficient cleavage activity. The results of the present work, in combination with those from previous reports, indicate that five critical functional groups are located within the tetrameric sequence G10A11U12G13. A preliminary model for the binding of a single magnesium cofactor to this portion of the sequence is proposed. In this model, the five critical functional groups interact with a partially hydrated magnesium cofactor, and the sixth coordination site remains open for complexing an oxygen from the scissile phosphodiester or for a similar interligand interaction involving a coordinated water molecule.