Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme

Abstract

In common with other self-cleaving RNAs, the lead-dependent ribozyme (leadzyme) undergoes dynamic fluctuations to a chemically activated conformation. We explored the connection between conformational dynamics and self-cleavage function in the leadzyme using a combination of NMR spin-relaxation analysis of ribose groups and conformational restriction via chemical modification. The functional studies were performed with a -methanocarbacytidine modification that prevents fluctuations to C2'-endo conformations while maintaining an intact 2'-hydroxyl nucleophile. Spin-relaxation data demonstrate that the active-site Cyt-6 undergoes conformational exchange attributed to sampling of a minor C2'-endo state with an exchange lifetime on the order of microseconds to tens of microseconds. A conformationally restricted species in which the fluctuations to the minor species are interrupted shows a drastic decrease in self-cleavage activity. Taken together, these data indicate that dynamic sampling of a minor species at the active site of this ribozyme, and likely of related naturally occurring motifs, is strongly coupled to catalytic function. The combination of NMR dynamics analysis with functional probing via conformational restriction is a general methodology for dissecting dynamics-function relationships in RNA.

RNA Dynamics by NMR Spectroscopy

“…Active site adenine is involved in protonation/deprotonation equilibrium, while cytosine residue samples minor C2'-endo state on at imescale of microseconds that is important for self-cleavage activity. [95,96] In line with CEST measurements, unfolded F À riboswitch was shown to sample an excited state that resembles folded riboswitch with k EX 121 s À1 and population of 10 %. [42] As econdary structural switch was observed for group Ii ntron ribozyme of T. thermophila with population of 3% and k EX of 423 s À1 ,w iths tructure resembling that of the native state.…”

MA’AT Analysis of Aldofuranosyl Rings: Unbiased Modeling of Conformational Equilibria and Dynamics in Solution

“…10 Presumably, the motional properties of the RNA backbone permit transient access to the 2 E form, which then allows productive attack of the O2′ nucleophile on phosphate to generate a penta-coordinate phosphate transition state and subsequent formation of a 2′,3′cyclic phosphate intermediate. 11 Although not often considered in the mechanism, the conformation of the β-D-ribofuranosyl ring may also affect the pK a of O2′H on the C residue, with a quasi-equatorial orientation of the C2′−O2′ bond in 2 E associated with a lower pK a than the quasi-axial orientation of the same bond in 3 E, possibly due to a shorter C−O bond in the former. 12 This reduction in pK a would promote hydroxyl hydrogen abstraction, thereby activating O2 as a nucleophile and promoting catalysis.…”

“…Which of these two states is preferred depends on the type of DNA or RNA duplex (A- or B-type) and on other structural factors (e.g., single- vs double-stranded; hybrid duplexes). These conformational options are exploited, for example, in protein-nucleic acid recognition and in the mechanism of intramolecular catalysis by ribozymes. , In the latter case, furanose ring conformational change in the C residue at the cleavage site is critical to proper alignment of its O2′ with an oxygen atom attached to phosphate on the adjacent C3′, the latter eventually becoming O5′ on the new 5′-end after cleavage (the two oxygens assume axial positions in the trigonal bipyramidal transition state of P). In this case, conversion of 3 E (N form, C3′- endo , favored in ground-state A-RNA) (Scheme ) to 2 E (S form, C2′- endo ) in the transition state is required for catalysis .…”

“…In this case, conversion of 3 E (N form, C3′- endo , favored in ground-state A-RNA) (Scheme ) to 2 E (S form, C2′- endo ) in the transition state is required for catalysis . Presumably, the motional properties of the RNA backbone permit transient access to the 2 E form, which then allows productive attack of the O2′ nucleophile on phosphate to generate a penta-coordinate phosphate transition state and subsequent formation of a 2′,3′-cyclic phosphate intermediate . Although not often considered in the mechanism, the conformation of the β- d -ribofuranosyl ring may also affect the p K a of O2′H on the C residue, with a quasi -equatorial orientation of the C2′–O2′ bond in 2 E associated with a lower p K a than the quasi -axial orientation of the same bond in 3 E , possibly due to a shorter C–O bond in the former .…”

Evaluation of the stereochemical quality of predicted RNA 3D models in the RNA-Puzzles submissions

“…Catalytic features, for instance, highlight that not only the model's geometry is important, but also its stereochemistry is an important factor as well. One example is the torsion-angle-based dependence between the active and nonactive conformation of base pairs in some ribozyme active sites ( White et al 2018 ). Moreover, self-cleaving ribozymes can provide another example, in which the correct description of phosphate backbone stereochemistry is critical to correctly assess the reaction pathways of these mechanisms ( Teplova et al 2020 ).…”