Analysis of the Conserved P9-G10.1 Metal-Binding Motif in Hammerhead Ribozymes with an Extra Nucleotide Inserted between A9 and G10.1 Residues

Abstract

Hammerhead ribozymes (Rz) have catalytically important tandem G:A pairs in the core region, and we recently demonstrated that the P9-G10.1 motif (a sheared-type G:A pair with a guanine residue on the 3' side of the adenine residue) with several flanking base pairs is sufficient for capture of divalent cations, such as Mg(2+) and Cd(2+) ions that are important to maintain full activities (Tanaka et al. J. Am. Chem. Soc. 2002, 124, 4595-4601; Tanaka et al. J. Am. Chem. Soc. 2004, 126, 744-752). We also found that mutant hammerhead ribozymes that have an additional G residue inserted between A9 and G10.1 residues (the metal-binding P9-G10.1 motif) have significant catalytic activities. In this study, we demonstrate that the hammerhead ribozymes are capable of maintaining the catalytically competent structure even when the tandem, sheared-type G:A pairs were perturbed by an insertion of an additional nucleotide, whereas the chirality of the phosphorothioate at the P9 position significantly influenced the enzymatic activity for both the natural and G-inserted ribozymes.

Catalytic Diversity of Extended Hammerhead Ribozymes

“…While the catalytic cores of nHH1 to nHH9 are not identical, the observed sequence variations at position 7 (U, G, or A) are not expected to significantly modify rates based on experiments with minimal hammerheads (43). A residue that is likely to be responsible for some of the catalytic diversity is the "bulged" nucleotide positioned between A9 and G10 (9), which substantially enhances cleavage of minimal hammerheads (10,44) and is present in the three fastest hammerheads studied here (nHH3, nHH5 and nHH8). Another source of catalytic diversity could be the varying sequence at the base pairs 1.1-1.2 and 2.1-2.2 which can modify cleavage rates in minimal hammerheads about 10-fold (45).…”

Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme

“…Analyses of the structure of hammerhead ribozymes and of the conformational changes caused by interactions with Mg 2+ ions have indicated that two major conformational changes occur: the formation of domain II, which is followed by the formation of domain I, as shown in Figure 1C (38,45,48,50,54). The formation of domain II results in coaxial stacking of helices II and III, induced by the binding of a higheraffinity Mg 2+ ion(s) to P9 phosphate and N7 of G 10.1 (P9/ G10.1) of the ribozyme-substrate complex (38,45,48,50,(54)(55)(56)(57)(58). The second transition is the formation of the catalytic domain with movement of stem I toward stem II, which is induced by the binding of a lower-affinity Mg 2+ ion(s).…”

Coupling of Fast and Slow Modes in the Reaction Pathway of the Minimal Hammerhead Ribozyme Cleavage

“…The conserved central bases, with few exceptions, are essential for ribozyme's catalytic activity (28). In terms of the structure (31)(32)(33)(34)(35)(36) and in terms of the cleavage chemistrykinetic studies (37)(38)(39), folding studies (29,(40)(41)(42)(43)(44), effects of ion binding and rescue (45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55), elemental substitution at the cleavage site (49,56), cross-linking (28), effects of protonation and proton transfer (57)(58)(59)(60), single molecule studies (42,(61)(62)(63), phosphoryl transfer mechanism (37,(64)(65)(66)(67)(68)(69)(70), alternative reaction mechanisms (71,72...…”