Authors:	Spenkuch, Felix Hinze, Gerald Kellner, Stefanie Kreutz, Christoph Micura, Ronald Basché, Thomas Helm, Mark
Title:	Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor
Online publication date:	10-Oct-2022
Year of first publication:	2014
Language:	english
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 K(d) 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.
DDC:	540 Chemie 540 Chemistry and allied sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 09 Chemie, Pharmazie u. Geowissensch.
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-7920
Version:	Published version
Publication type:	Zeitschriftenaufsatz
License:	CC BY
Information on rights of use:	https://creativecommons.org/licenses/by/4.0/
Journal:	Nucleic acids research 42 20
Pages or article number:	12735 12745
Publisher:	Oxford Univ. Press
Publisher place:	Oxford
Issue date:	2014
ISSN:	0305-1048 1362-4962
Publisher URL:	http://dx.doi.org/10.1093/nar/gku908
Publisher DOI:	10.1093/nar/gku908
Appears in collections:	DFG-OA-Publizieren (2012 - 2017)