Topological rearrangement yields structural stabilization and interhelical distance constraints in the Kin.46...

The Kin.46 ribozyme catalyzes transfer of the gamma (thio)phosphoryl group of ATP (or ATPgammaS) to the ribozyme's 5' hydroxyl. Single-turnover catalytic activities of topologically rearranged versions of Kin.46 were studied to gain insight into its overall tertiary architecture. The distal ends of stems P3 and P4 were tethered through a single-stranded connection domain that altered the interhelical connectivity. The shortest linkers interfered with catalysis, while seven or more nucleotides (nt) in the linker allowed near-normal catalytic rates, suggesting that a distance of roughly 25-35 A optimally separates the termini of these helices. Activity was maximal when the tether contained 15 nt, at which point the k(cat) (0.016 min(-1)) and Km (1.2 mM) values were identical to those of a nontethered control. The presence of the tether alters Mg(2+) dependence, in that Mg2+ binding appears to be more cooperative in the tethered ribozyme (Hill coefficient 1.4-1.8 versus 0.8 for the nontethered ribozyme). Binding affinity for the ATPgammaS substrate increases at elevated concentrations of Mg2+, particularly for the tethered ribozyme. The tethered ribozyme displays significantly enhanced thermal stability, with a maximum initial velocity (0.126 min(-1)) at 60 degrees C, whereas the nontethered ribozyme has a lower maximum initial velocity (0.051 min(-1)) at 50 degrees C. The tether also significantly reduces the apparent entropy of activation. Both of these effects can be understood in terms of stabilization of the ribozyme in a conformation that is on-path with respect to catalysis, and in terms of facilitating formation of the allosteric activation helix P4.