title: Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor.
abstract: The interest in RNA modification enzymes surges due to their involvement in epigenetic phenomena. Here we present a particularly informative approach to investigate the interaction of dye-labeled RNA with modification enzymes. We investigated pseudouridine (Ψ) synthase TruB interacting with an alleged suicide substrate RNA containing 5-fluorouridine (5FU). A longstanding dogma, stipulating formation of a stable covalent complex was challenged by discrepancies between the time scale of complex formation and enzymatic turnover. Instead of classic mutagenesis, we used differentially positioned fluorescent labels to modulate substrate properties in a range of enzymatic conversion between 6% and 99%. Despite this variegation, formation of SDS-stable complexes occurred instantaneously for all 5FU-substrates. Protein binding was investigated by advanced fluorescence spectroscopy allowing unprecedented simultaneous detection of change in fluorescence lifetime, anisotropy decay, as well as emission and excitation maxima. Determination of Kd values showed that introduction of 5FU into the RNA substrate increased protein affinity by 14× at most. Finally, competition experiments demonstrated reversibility of complex formation for 5FU-RNA. Our results lead us to conclude that the hitherto postulated long-term covalent interaction of TruB with 5FU tRNA is based on the interpretation of artifacts. This is likely true for the entire class of pseudouridine synthases.
authors.affiliation: Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55128 Mainz, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Spenkuch
authors.affiliation: Institute of Physical Chemistry, University of Mainz, Duesbergweg 10-14, D-55128 Mainz, Germany.
authors.forename: Gerald
authors.initials: G
authors.lastname: Hinze
authors.affiliation: Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55128 Mainz, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Kellner
authors.affiliation: Institute of Organic Chemistry, Center for Molecular Biosciences (CMBI), University of Innsbruck, Innrain 52A, A-60230 Innsbruck, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Kreutz
authors.affiliation: Institute of Organic Chemistry, Center for Chemistry and Biomedicine - CCB, University of Innsbruck, Innrain 80/82, A-60230 Innsbruck, Austria.
authors.forename: Ronald
authors.initials: R
authors.lastname: Micura
authors.affiliation: Institute of Physical Chemistry, University of Mainz, Duesbergweg 10-14, D-55128 Mainz, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Basché
authors.affiliation: Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55128 Mainz, Germany mhelm@uni-mainz.de.
authors.forename: Mark
authors.initials: M
authors.lastname: Helm
mesh_terms: D016477:Artifacts; D002853:Chromatography, Liquid; D005456:Fluorescent Dyes; D019751:Intramolecular Transferases / Q000378:metabolism*; D011485:Protein Binding; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D053719:Tandem Mass Spectrometry; D014529:Uridine / Q000031:analogs & derivatives*
chemical_list: D005456:Fluorescent Dyes; C001943:5-fluorouridine; D012343:RNA, Transfer; D019751:Intramolecular Transferases; C096995:pseudouridine synthases; D014529:Uridine