Critical nature of a specific uridine O-2-carbonyl for cleavage by the hammerhead ribozyme

Three modified hammerhead ribozyme/substrate complexes have been prepared i n which individual uridine O-2-carbonyls have been eliminated. The modified complexes were chemically synthesized with the substitution of a single 2- pyridone (2P) base analogue for residues U-4, U-7, and U-16.1. Steady-state kinetic analyses indicate that the cleavage efficiencies for the U-7 and U -16.1 complexes were not significantly reduced relative to the native compl ex as measured by k(cat)/K-M. The cleavage efficiency for the 2P(4) complex , with the analogue present within the uridine loop, was reduced by greater than 2 orders of magnitude. This significant reduction in catalytic effici ency was due primarily to a decrease in k(cat). The pH vs cleavage rate pro file suggests that the O-2-carbonyl of the U-4 residue of the hammerhead co mplex is critical for transition state stabilization and efficient cleavage activity. The results of a Mg2+ rescue assay do not implicate the O-2-carb onyl of U-4 in an interaction with a divalent metal ion. In addition, the r esults of a ribozyme folding assay suggest that the presence of the 2P(4) w ithin the uridine loop does not alter the folding pathway (relative to the native sequence) both in the absence and in the presence of Mg2+. The O-2-c arbonyl of U-4 appears oriented toward the interior of the catalytic pocket where it may be involved in a critical hydrogen bonding interaction necess ary for transition state stabilization.