Kinetic analysis of pseudouridine formation in tRNA by archaeal Cbf5 and Pus10

Abstract

Pseudouridines (Ψ), the most common modifications in RNA, are formed by stand-alone Ψ synthases in all organisms. In addition, archaea and eukaryotes use H/ACA small ribonucleoproteins for pseudouridylation. Cbf5, the catalytic component of these complexes, can also introduce Ψ55 in archaeal tRNAs in a guide RNA-independent manner. Here, kinetic and thermodynamic analyses revealed that both Pyrococcus furiosus Nop10 and Gar1 proteins enhance the catalytic ability of Cbf5 and increase its affinity for tRNA. Pus10, representing a novel Ψ synthase family, is the in vivo archaeal tRNA Ψ55 synthase. Characterization of several Pus10 variants demonstrated the importance of the thumb loop for catalysis, a potential role of the THUMP domain in tRNA binding and a new catalytic arginine which may flip the target uridine into Pus10’s active site. The quantitative characterization of the archaeal pseudouridine synthases Cbf5 and Pus10 reported here sheds light on their cellular roles in RNA modification.