title: tRNA binding, positioning, and modification by the pseudouridine synthase Pus10.
abstract: Pus10 is the most recently identified pseudouridine synthase found in archaea and higher eukaryotes. It modifies uridine 55 in the TΨC arm of tRNAs. Here, we report the first quantitative biochemical analysis of tRNA binding and pseudouridine formation by Pyrococcus furiosus Pus10. The affinity of Pus10 for both substrate and product tRNA is high (Kd of 30nM), and product formation occurs with a Km of 400nM and a kcat of 0.9s(-1). Site-directed mutagenesis was used to demonstrate that the thumb loop in the catalytic domain is important for efficient catalysis; we propose that the thumb loop positions the tRNA within the active site. Furthermore, a new catalytic arginine residue was identified (arginine 208), which is likely responsible for triggering flipping of the target uridine into the active site of Pus10. Lastly, our data support the proposal that the THUMP-containing domain, found in the N-terminus of Pus10, contributes to binding of tRNA. Together, our findings are consistent with the hypothesis that tRNA binding by Pus10 occurs through an induced-fit mechanism, which is a prerequisite for efficient pseudouridine formation.
authors.affiliation: Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive, Lethbridge, AB, T1K 3M4, Canada. Electronic address: rajashekhar.kamalamp@uleth.ca.
authors.forename: Rajashekhar
authors.initials: R
authors.lastname: Kamalampeta
authors.forename: Laura C
authors.initials: LC
authors.lastname: Keffer-Wilkes
authors.forename: Ute
authors.initials: U
authors.lastname: Kothe
mesh_terms: D002384:Catalysis; D002942:Circular Dichroism; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011560:Pseudouridine / Q000096:biosynthesis; D020027:Pyrococcus furiosus / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011560:Pseudouridine; D012343:RNA, Transfer; D006836:Hydro-Lyases; C022479:pseudouridylate synthetase
keywords: Michaelis–Menten kinetics; RNA binding; RNA modification; tRNA; thumb loop