Conformational Dynamics of Self-thiophosphorylating RNA

Abstract

The Kin.46 kinase ribozyme was previously selected from a random-sequence RNA library that was loosely based on the sequence of a known ATP aptamer, based on its ability to catalyze transfer of the thiophosphate from ATPgS to its own 5' hydroxyl end. The reaction requires an oligonucleotide effector that is complementary to the 3' end of the ribozyme and that served as reverse transcription primer during the amplification steps of the original selection for activity (Fig. 1). Omitting the oligonucleotide reduces the observed catalytic rate constant (kobs) by 10 3 to 10-fold, indicating that the oligo acts as an allosteric effector that is necessary for full catalytic activity. The activator helix is separated from the substrate-binding internal guide sequence by a 5nt “linker” that appears to form long-range baseparing interaction with nucleotides within the catalytic core (S. Rhee, unpublished results). Binding of small ligands, such as metal ions and ATP, can also potentially influence the folded structure of the RNA. Divalent metal ions are well known for their abilities to stabilize nucleic acid secondary and tertiary structures, and for their potential catalytic roles in ribozyme-catalyzed reactions. For example, we recently found that topologically rearranged versions of Kin.46 exhibit markedly altered Mg dependence. Similarly, nucleotide binding by aptamers nucleates formation of the folded structure and induces order in the NTP binding pocket (adaptive binding). ATP recognition by Kin.46 is clearly distinct from ATP recognition by the original aptamer; key nucleotides required for ATP recognition are mutated in Kin.46, and the values of Km ATPgS and Km ATP for Kin.46 (~3 mM) are >10 times higher than Kd ATP for the aptamer (~1 μM). In this work, fluorescence emission from cy5-labeled ribozymes was monitored in order to investigate conformational dynamics as a function of ATP concentration and Mg ion. Truncated versions of Kin.46 ribozyme were obtained by internal cleavage or deletions (Fig. 1). Ribozyme130 was derived from the Kin.46 by just cleaving the most right-side loop, ribozyme119 was obtained by deletion of the loop and