An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Abstract

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2 ′ -and 3 ′ -deoxy (-H) and −amino (-NH 2 ) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3 ′ -OH making a nonproductive interaction with an active site metal ion termed M A and with the adjacent 2 ′ -OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed M C , to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function.

Lessons From Catalysis by Rna Enzymes

“…Consider the catalytic interactions and active site architecture depicted in Figure 1C-E for the group I ribozyme, which are supported by a substantial interplay of functional and structural results [34][35][36]. One Mg 2+ ion activates the guanosine (G) nucleophile and the other Mg 2+ ion stabilizes charge development on the leaving group oxygen, and both Mg 2+ ions interact with a nonbridging phosphoryl oxygen atom, likely making favorable electrostatic interactions and providing a template for the transition state.…”

“…C-E) Model of the group I ribozyme active site. C) Transition state model derived from biochemical and structural data (see [36] and references therein). Closed circles and hatched lines represent metal ion interactions and hydrogen bonds, respectively.…”

“…Partialnegative charges in are represented by "δ − ." D) Model of the ground state ESG complex [36]. E) Model of the network of interactions within the active site of the ESG complex of the group I ribozyme [36].…”

“…D) Model of the ground state ESG complex [36]. E) Model of the network of interactions within the active site of the ESG complex of the group I ribozyme [36]. Atoms highlighted in magenta contact M A , M C , and G. For The location of this network within the overall structure of the Azoarcus group I ribozyme is shown on the right.…”

Differential Assembly of Catalytic Interactions within the Conserved Active Sites of Two Ribozymes

“…This ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine cofactor (G) [ 17 ]. G binding to the Tetrahymena ribozyme is extraordinarily slow, (~10 5 M -1 min -1 [ 8 ]), orders of magnitude below the diffusion limit, indicating that the G binding site exists predominantly in one or more states that do not allow G binding [ 8 , 18 , 19 ], and additional experiments [ 8 ] provided evidence for a transient opening, with a rate constant of ~10 2 –10 3 s -1 , to a state that is competent to bind G.…”