Topological rearrangement yields structural stabilization and interhelical distance constraints in the Kin.46 self-phosphorylating ribozyme

Abstract

The Kin.46 ribozyme catalyzes transfer of the gamma (thio)phosphoryl group of ATP (or ATPγS) 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 Å optimally separates the termini of these helices. Activity was maximal when the tether contained 15 nt, at which point the kcat (0.016 min -1) and Km (1.2 mM) values were identical to those of a nontethered control. The presence of the tether alters Mg2+ 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 ATPgS 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°C, whereas the nontethered ribozyme has a lower maximum initial velocity (0.051 min -1) at 50°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. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 RNA Society.