Structural dynamics govern substrate recruitment and catalytic turnover in H/ACA RNP pseudouridylation

H/ACA ribonucleoproteins catalyse the sequence-dependent pseudouridylation of ribosomal and spliceosomal RNAs. Here, we reconstitute site-specifically fluorophore labelled H/ACA complexes and analyse their structural dynamics using single-molecule FRET spectroscopy. Our results show that the guide RNA is distorted into a substrate-binding competent conformation by specific protein interactions. Analysis of the reaction pathway using atomic mutagenesis establishes a new model how individual protein domains contribute to catalysis. Taken together, these results identify and characterize individual roles for all accessory proteins on the assembly and function of H/ACA RNPs.

Automated summary

The study reconstructed site-specifically fluorophore labelled H/ACA complexes and analyzed their structural dynamics using single-molecule FRET spectroscopy. Distortion of the guide RNA into a substrate-binding competent conformation by specific protein interactions was observed, and a new model explaining the contribution of individual protein domains to catalysis was established through atomic mutagenesis. These results identify and characterize individual roles for all accessory proteins in the assembly and function of H/ACA RNPs.