IMPORTANCE OF SPECIFIC ADENOSINE N-3-NITROGENS FOR EFFICIENT CLEAVAGEBY A HAMMERHEAD RIBOZYME

Five modified hammerhead ribozyme/substrate complexes have been prepar ed in which individual adenosine N-3-nitrogens have been excised and r eplaced with carbon. The modified complexes were chemically synthesize d with the substitution of a single 3-deazaadenosine (c(3)A) base anal ogue for residues A(6), A(9), A(13), A(14), or A(15.1). Steady-state k inetic analyses indicate that the cleavage efficiencies, as measured b y k(cat)K(M), for the c(3)A(6), c(3)A(9), and c(3)A(14) complexes were only marginally reduced (less than or equal to 5-fold) relative to th e native complex. By comparison, the cleavage efficiencies for the c(3 )A(13) and c(3)A(15.1) complexes were reduced by 9-fold and 55-fold, r espectively. These reductions in cleavage efficiency are primarily a r esult of lower k(cat) values. Profiles of pH and cleavage rate suggest that the chemical cleavage step is the rate-limiting reaction for the se complexes. These results suggest that the N-3-nitrogen of the A(13) residue and particularly the A(15.1) residue in the hammerhead ribozy me/substrate complex are critical for transition state stabilization a nd efficient cleavage activity. We have additionally compared the loca tions of thee critical functional groups, as well as those identified from other studies, with recent crystallographic analyses. In some cas es, the critical functional groups are clustered around proposed metal binding sites and may reflect functional groups critical for binding the metal cofactor. In other cases, clusters of functional groups may form a network of hydrogen bonds necessary for transition state stabil ization.