Structure–function relationships in the hammerhead ribozyme probed by base rescue

Abstract

We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc Nat Acad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structurefunction studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.

A Role for the 2′ OH of Peptidyl-tRNA Substrate in Peptide Release on the Ribosome Revealed through RF-Mediated Rescue

“…We observed similar, albeit more modest, effects with the Q235G and Q235C variants. Rescue activity has been observed previously in other ribozymes, usually by adding free bases to an abasic site, and can be used to probe ribozyme mechanism (Peracchi et al, 1996; Peracchi et al, 1998). Interestingly, here we observe rescue of an RNA mutation by a protein moiety.…”

Chemical rescue, multiple ionizable groups, and general acid–base catalysis in the HDV genomic ribozyme

“…This approach to study an enzyme's catalytic mechanism was first used by Toney and Kirsch (1989) to evaluate general acid-base catalysis in aspartate aminotransferase. Chemical (base) rescue has also been used in structurefunction studies of the hammerhead ribozyme containing abasic residues in the conserved core region to probe and identify transition state interactions (Peracchi et al 1996(Peracchi et al , 1998. More recently, chemical rescue has been used with the hairpin ribozyme in defining the catalytic roles of the G8 and A38 side chains (Lebruska et al 2002;Kuzmin et al 2004Kuzmin et al , 2005.…”

Challenges in Enzyme Mechanism and Energetics

“…It is of course highly unlikely that each of these ribozyme residues plays a direct role in catalysis; nor do the modifications significantly affect substrate binding (68,69). What then is the origin of the large and distributed mutational effects in the hammerhead core?…”