A Core Folding Model for Catalysis by the Hammerhead Ribozyme Accounts for Its Extraordinary Sensitivity to Abasic Mutations

Abstract

Introducing abasic nucleotides at each of 13 positions in the conserved core of the hammerhead ribozyme causes a large decrease in the extent of catalysis [Peracchi, A., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 11522]. This extreme sensitivity to structural defects is in contrast to the behavior of protein enzymes and larger ribozymes. Several additional differences in the behavior of the hammerhead relative to that of protein enzymes and larger ribozymes are described herein. The deleterious effects of the abasic mutations are not relieved by lowering the temperature, by increasing the concentration of monovalent or divalent metal ions, or by adding polyamines, in contrast to effects observed with protein enzymes and large RNA enzymes. In addition, the abasic mutations do not significantly weaken substrate binding. These results and previous observations are all accounted for by a "core folding" model in which the stable ground state structure of the hammerhead ribozyme complexed with the substrate is a partially folded state that must undergo an additional folding event to achieve its catalytic conformation. We propose that the peculiar behavior of the hammerhead arises because the limited structural interconnections in a small RNA enzyme do not allow the ground state to stably adopt the catalytic conformation; within the globally folded catalytic conformation, limited structural interconnections may further impair catalysis by hampering the precise alignment of active site functional groups. This behavior represents a basic manifestation of the well-recognized interconnection between folding and catalysis.The hammerhead (Figure 1) is the smallest known naturally occurring ribozyme (see refs 3-5 for review), and the only one for which a complete three-dimensional structure is known to atomic resolution (6, 7). Previously, we began to address the structural basis of hammerhead catalysis through a "subtraction mutagenesis" approach, in which each residue in the conserved core was individually replaced by an abasic nucleotide analogue (Chart 1) (8,9). Removing individual bases from DNA duplexes induces local, context-dependent rearrangements (10-16), and removing large amino acid side chains from protein cores (e.g., via alanine scanning mutagenesis) can result in various rearrangements or can leave a cavity within the core (17)(18)(19). Analogously, abasic mutagenesis in the hammerhead core could reveal how this ribozyme responds functionally to the introduction of structural defects and to changes in local packing.Nearly all of the 13 positions in the hammerhead ribozyme core that were substituted with an abasic residue gave large decreases in the extent of catalysis (8) ( Figure 1B). In contrast, mutations at only a small subset of "catalytic" residues in proteins typically yield a large decrease in activity (see below). The characterization of the abasic hammerhead variants reported herein was carried out in an effort to understand why so many abasic substitutions in the hammerhead produce s...

Significant activity of a modified ribozyme with N7‐deazaguanine at G10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

“…However, the extent of the reduction (30-fold) caused by the removal of N7 of G 10.1 was much smaller than that (1000-fold reduction) caused by the introduction of the Rp-phosphorothioate at the P9 position (Peracchi et al 1997). This result suggests that, even though binding of Mg 2 to N7 of G 10.1 is catalytically important (Peracchi et al 1998;Wang et al 1999), it is not absolutely indispensable. Table 1.…”

“…In some cases, the metal ion can be directly coordinated to N7 of G 10.1 (Scott et al 1996;Murray et al 1998a, b) and the sequence motif in such cases is known to be a metal-binding motif (Baeyens et al 1996;Peracchi et al 1998). In biochemical studies, speci®c phosphodiester bonds that are critical for catalysis by hammerhead ribozymes were identi®ed by the pioneering work of Uhlenbeck's group (Ruffner & Y Nakamatsu et al 608 Genes to Cells (2000) 5, 603±612 q Blackwell Science Limited Figure 4 Possible mechanism for the cleavage of S13 that is catalysed by 7-deaza-R34 as the concentration of LaCl 3 is increased in the presence of a ®xed concentration of MgCl 2 .…”

Efficient Ligation of the Schistosoma Hammerhead Ribozyme

“…Peracchi et al proposed a model where the hammerhead ribozyme exists in multiple conformations in solution but only a subset of these conformations are catalytically active (59). Nelson & Uhlenbeck extended this model on the basis of the crystal structure of the Schistosoma hammerhead where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules, resulting in higher catalytic activity for the ribozyme (30).…”

The P5abc Peripheral Element Facilitates Preorganization of the Tetrahymena Group I Ribozyme for Catalysis

“…The dearth of structural information in the primary sequence of RNA (4; also see the Introduction) may render it difficult to specifically stabilize a functional structure without the preorganization afforded by interconnected elements outside the catalytic core. The folding of the hammerhead ribozyme, which lacks peripheral sequences, appears to be so tenuous that mutation of almost any of the core residues is sufficient to reduce activity by more than 10 4 -fold (74). Further, in contrast to the preorganization of the core of the Tetrahymena ribozyme suggested by structural and functional data (15,17,18,65), the hammerhead ribozyme lacks an active site crevice and seemingly requires a substantial conformational change prior to catalysis (75,76).…”