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 North-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.