IMPORTANCE OF SPECIFIC GUANOSINE N7-NITROGENS AND PURINE AMINO-GROUPSFOR EFFICIENT CLEAVAGE BY A HAMMERHEAD RIBOZYME

Seven modified hammerhead ribozyme/substrate complexes have been prepa red in which individual purine nitrogens, the guanine N7-, the guanine N2-, or the adenine N6-nitrogen, have been excised. The modified comp lexes were chemically synthesized with the substitution of a single 7- deazaguanosine (c7G), inosine (I), or nebularine (purine riboside, P) base analogue as appropriate for residues G5, G8, G12, A13, A14, or A1 5. Two of the base analogues, c7G5 and c7G8, occur in a 19-mer ribozym e, while the remaining three residues are present in a 24-mer substrat e. Under stoichiometric conditions, four of the complexes, G5c7G, G8c7 G, G12c7G, and A14P, are cleaved with relatively little change in rate when compared with the native complex. Two complexes, A13P and A15P, are cleaved some 6-8-fold slower than the native complex, while the G1 2I complex is reduced in rate by 50-fold. Steady-state kinetic analyse s indicate that the cleavage efficiencies, as measured by k(cat)/K(M) values, for the G5c7G, G8c7G, and G12c7G complexes are only marginally reduced relative to the native complex. The values for the A13P, A14P , and A15P complexes are reduced by 25-, 15-, and 60-fold, respectivel y. These reductions in cleavage efficiency are primarily a result of l ower k(cat) values. By comparison, the k(cat)/K(M) value for the G12I complex is decreased 450-fold relative to the native complex and is ch aracterized by an 8-fold increase in K(M) and a k(cat) value that is r educed nearly 60-fold. These results indicate that the N2-amino group of G12 in the hammerhead ribozyme/substrate complex is critical for ef ficient cleavage activity. The loss of the amino groups from A13, A14, and A15 impact the cleavage efficiency, but these complexes remain at least 30-fold more active than the G12I complex. The N7-nitrogens of the conserved guanine residues do not appear to take part in any criti cal hydrogen-bonding/metal-ion-coordinating interactions or to have an y other significant role in the observed catalytic transesterification reaction.