Gutenberg Open Science: Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor

Please use this identifier to cite or link to this item: http://doi.org/10.25358/openscience-7920

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DC Field	Value	Language
dc.contributor.author	Spenkuch, Felix	-
dc.contributor.author	Hinze, Gerald	-
dc.contributor.author	Kellner, Stefanie	-
dc.contributor.author	Kreutz, Christoph	-
dc.contributor.author	Micura, Ronald	-
dc.contributor.author	Basché, Thomas	-
dc.contributor.author	Helm, Mark	-
dc.date.accessioned	2022-10-10T07:53:37Z	-
dc.date.available	2022-10-10T07:53:37Z	-
dc.date.issued	2014
dc.identifier.uri	https://openscience.ub.uni-mainz.de/handle/20.500.12030/7935	-
dc.description.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.	en_GB
dc.description.sponsorship	DFG, Open Access-Publizieren Universität Mainz / Universitätsmedizin	de
dc.language.iso	eng	de
dc.rights	CC BY	*
dc.rights.uri	https://creativecommons.org/licenses/by/4.0/	*
dc.subject.ddc	540 Chemie	de_DE
dc.subject.ddc	540 Chemistry and allied sciences	en_GB
dc.title	Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor	en_GB
dc.type	Zeitschriftenaufsatz	de
dc.identifier.doi	http://doi.org/10.25358/openscience-7920	-
jgu.type.dinitype	article	en_GB
jgu.type.version	Published version	de
jgu.type.resource	Text	de
jgu.organisation.department	FB 09 Chemie, Pharmazie u. Geowissensch.	de
jgu.organisation.number	7950	-
jgu.organisation.name	Johannes Gutenberg-Universität Mainz	-
jgu.rights.accessrights	openAccess	-
jgu.journal.title	Nucleic acids research	de
jgu.journal.volume	42	de
jgu.journal.issue	20	de
jgu.pages.start	12735	de
jgu.pages.end	12745	de
jgu.publisher.year	2014	-
jgu.publisher.name	Oxford Univ. Press	de
jgu.publisher.place	Oxford	de
jgu.publisher.uri	http://dx.doi.org/10.1093/nar/gku908	de
jgu.publisher.issn	0305-1048	de
jgu.publisher.issn	1362-4962	de
jgu.organisation.place	Mainz	-
jgu.subject.ddccode	540	de
opus.date.modified	2018-08-14T09:14:00Z
opus.subject.dfgcode	00-000
opus.organisation.string	FB 09: Chemie, Pharmazie und Geowissenschaften: Institut für Physikalische Chemie	de_DE
opus.organisation.string	FB 09: Chemie, Pharmazie und Geowissenschaften: Institut für Pharmazie	de_DE
opus.identifier.opusid	58415
opus.institute.number	0906
opus.institute.number	0908
opus.metadataonly	false
opus.type.contenttype	Keine	de_DE
opus.type.contenttype	None	en_EN
opus.affiliated	Hinze, Gerald
opus.affiliated	Basché, Thomas
opus.affiliated	Helm, Mark
jgu.publisher.doi	10.1093/nar/gku908	de
jgu.organisation.ror	https://ror.org/023b0x485	-
Appears in collections:	DFG-OA-Publizieren (2012 - 2017)

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