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

Abstract

Molecular recognition is central to biology and a critical aspect of RNA function. Yet structured RNAs typically lack the preorganization needed for strong binding and precise positioning. A striking example is the group I ribozyme from Tetrahymena, which binds its guanosine substrate (G) orders of magnitude slower than diffusion. Binding of G is also thermodynamically coupled to binding of the oligonucleotide substrate (S) and further work has shown that the transition from E•G to E•S•G accompanies a conformational change that allows G to make the active site interactions required for catalysis. The group I ribozyme from Azoarcus has a similarly slow association rate but lacks the coupled binding observed for the Tetrahymena ribozyme. Here we test, using G analogs and metal ion rescue experiments, whether this absence of coupling arises from a higher degree of preorganization within the Azoarcus active site. Our results suggest that the Azoarcus ribozyme forms cognate catalytic metal ion interactions with G in the E•G complex, interactions that are absent in the Tetrahymena E•G complex. Thus, RNAs that share highly similar active site architectures and catalyze the same reactions can differ in the assembly of transition state interactions. More generally, an ability to readily access distinct local conformational states may have facilitated the evolutionary exploration needed to attain RNA machines that carry out complex, multi-step processes.

Fig 1. The group I ribozyme reaction.

(A) General framework for the group I ribozyme reaction (left) presenting equilibrium dissociation constants of the G and S substrates to the Tetrahymena (middle, [20, 22]) and Azoarcus (right, [23]) ribozymes. (B) Model for the assembly of catalytic metal ion interactions with M_A (blue) and M_C (orange) in the Tetrahymena group I ribozyme active site [19, 21, 24]. Closed dots and hatched lines represent metal ion interaction and hydrogen bonds, respectively. Partial negative charges are represented by ‘δ^-’. Prior data suggest that a third metal ion, M_B, contacts the G 3′-oxygen atom instead of M_C in the transition state [25] but this interaction is likely an artifact from functional experiments, as described in the Discussion and elsewhere [19]. For simplicity, we show M_C contacting the G 3′-oxygen atom in the transition state.

Fig 2. Effects of 2′- and 3′-modifications on G binding to the Azoarcus ribozyme.

Equilibrium binding constants (K_ds) were obtained as described in Materials and Methods. (A,C) The effects of 2′-NH₂ and 2′-H substitutions on G binding to E (A) and ES (C). Values for binding of G represent the mean equilibrium binding constant from several independent measurements at different pH values at 15 mM Mg²⁺ in the absence or presence of 10 mM Mn²⁺ (S1 and S2 Figs). Values for binding of G(2′NH₂) are derived from the fit to the model shown in S4–S6 Figs. The value for binding of G(2′NH₂) to ES in the presence of Mn²⁺ represents the mean equilibrium binding constant from several independent measurements (S7 Fig). The fits to determine the K_d for G(2′H) are shown in S10 Fig. (B,D) The effects of 3′-NH₂ and 3′-H substitutions on G binding to E (B) and ES (D). Values for binding of G at 15 mM Mg²⁺ were obtained as described above. Values for binding of G(3′NH₂) were derived from the fit to the model shown in S8 and S9 Figs. Measurements were made at 100 mM Mg²⁺ to attenuate strong binding of G(3′N${\text{H}}_3^{+}$) (Materials and Methods). The arrows for the binding constants of G(3′NH₂) denote that the observed K_ds are lower limits (S8 and S9 Figs). Raising the Mg²⁺ concentration to 100 mM had a negligible effect on binding of G (S3 Fig), so that the affinities of G(3′NH₂) can be compared with binding affinities for G at 15 mM Mg²⁺. The fits to determine the K_d for G(3′H) are shown in S10 Fig.

Fig 3. Conformational landscapes of the G binding site.

Qualitative energy landscapes of the G binding sites of E (A) and the EG (B) and ESG complex (C), for the Tetrahymena and Azoarcus ribozymes (black and red lines, respectively). The G binding site is shown as a surface representation with or without bound G (green). G is also represented in stick form in panels B and C to highlight active site metal ion interactions in the inactive and active states [19]. The energy landscape and the accompanying structures were constructed as described in Materials and Methods.