UNUSUAL DYNAMICS AND PK(A) SHIFT AT THE ACTIVE-SITE OF A LEAD-DEPENDENT RIBOZYME

Heteronuclear NMR spectroscopy has been used to probe the structure an d dynamics of a lead-dependent ribozyme known as the leadzyme. The pK( a)'s for all adenine bases in the leadzyme were measured and vary from <3.1 to 6.5. The adenines with the lowest pK(a)'s are involved in Wat son-Crick base pairs, and the adenine with the highest pK(a) is though t to form an AH(+)-C wobble base pair at the active site of the leadzy me. The potential structural and functional significance associated wi th perturbed pK(a)'s in ribozymes is discussed. NMR studies of 5' AMP, 5' CMP, 5' GMP, and 5' UMP were also used to identify the C-13 resona nces that represent the best probes of base protonation for all four b ases in RNA. The line width of the C2 resonance of the adenine with a pK(a) of 6.5 was analyzed to probe the dynamics of the catalytically a ctive internal loop in the leadzyme. These results showed that this ad enine is undergoing a chemical exchange process with a lifetime of sim ilar to 30 mu s and demonstrate rapid dynamics at the active site of t his ribozyme. Thus C-13 NMR can be used to probe dynamic processes in RNA oligomers.