Formation of a stalled early intermediate of pseudouridine synthesis monitored by real-time FRET
- Publikationstyp:
- Zeitschriftenaufsatz
- Metadaten:
-
- Autoren
- Martin Hengesbach
- Felix Voigts-Hoffmann
- Benjamin Hofmann
- Mark Helm
- Autoren-URL
- https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=fis-test-1&SrcAuth=WosAPI&KeyUT=WOS:000274707500015&DestLinkType=FullRecord&DestApp=WOS_CPL
- DOI
- 10.1261/rna.1832510
- eISSN
- 1469-9001
- Externe Identifier
- Clarivate Analytics Document Solution ID: 557YV
- PubMed Identifier: 20106954
- ISSN
- 1355-8382
- Ausgabe der Veröffentlichung
- 3
- Zeitschrift
- RNA
- Schlüsselwörter
- RNA modification
- pseudouridine synthase
- FRET
- 5-fluorouridine
- Pus inhibition
- tRNA structure
- Paginierung
- 610 - 620
- Datum der Veröffentlichung
- 2010
- Status
- Published
- Titel
- Formation of a stalled early intermediate of pseudouridine synthesis monitored by real-time FRET
- Sub types
- Article
- Ausgabe der Zeitschrift
- 16
Datenquelle: Web of Science (Lite)
- Andere Metadatenquellen:
-
- Abstract
- <jats:p>Pseudouridine is the most abundant of more than 100 chemically distinct natural ribonucleotide modifications. Its synthesis consists of an isomerization reaction of a uridine residue in the RNA chain and is catalyzed by pseudouridine synthases. The unusual reaction mechanism has become the object of renewed research effort, frequently involving replacement of the substrate uridines with 5-fluorouracil (f<jats:sup>5</jats:sup>U). f<jats:sup>5</jats:sup>U is known to be a potent inhibitor of pseudouridine synthase activity, but the effect varies among the target pseudouridine synthases. Derivatives of f<jats:sup>5</jats:sup>U have previously been detected, which are thought to be either hydrolysis products of covalent enzyme-RNA adducts, or isomerization intermediates. Here we describe the interaction of pseudouridine synthase 1 (Pus1p) with f<jats:sup>5</jats:sup>U-containing tRNA. The interaction described is specific to Pus1p and position 27 in the tRNA anticodon stem, but the enzyme neither forms a covalent adduct nor stalls at a previously identified reaction intermediate of f<jats:sup>5</jats:sup>U. The f<jats:sup>5</jats:sup>U27 residue, as analyzed by a DNAzyme-based assay using TLC and mass spectrometry, displayed physicochemical properties unaltered by the reversible interaction with Pus1p. Thus, Pus1p binds an f<jats:sup>5</jats:sup>U-containing substrate, but, in contrast to other pseudouridine synthases, leaves the chemical structure of f<jats:sup>5</jats:sup>U unchanged. The specific, but nonproductive, interaction demonstrated here thus constitutes an intermediate of Pus turnover, stalled by the presence of f<jats:sup>5</jats:sup>U in an early state of catalysis. Observation of the interaction of Pus1p with fluorescence-labeled tRNA by a real-time readout of fluorescence anisotropy and FRET revealed significant structural distortion of f<jats:sup>5</jats:sup>U-tRNA structure in the stalled intermediate state of pseudouridine catalysis.</jats:p>
- Autoren
- Martin Hengesbach
- Felix Voigts-Hoffmann
- Benjamin Hofmann
- Mark Helm
- DOI
- 10.1261/rna.1832510
- eISSN
- 1469-9001
- ISSN
- 1355-8382
- Ausgabe der Veröffentlichung
- 3
- Zeitschrift
- RNA
- Sprache
- en
- Online publication date
- 2010
- Paginierung
- 610 - 620
- Datum der Veröffentlichung
- 2010
- Status
- Published
- Herausgeber
- Cold Spring Harbor Laboratory
- Herausgeber URL
- http://dx.doi.org/10.1261/rna.1832510
- Datum der Datenerfassung
- 2021
- Titel
- Formation of a stalled early intermediate of pseudouridine synthesis monitored by real-time FRET
- Ausgabe der Zeitschrift
- 16
Datenquelle: Crossref
- Abstract
- Pseudouridine is the most abundant of more than 100 chemically distinct natural ribonucleotide modifications. Its synthesis consists of an isomerization reaction of a uridine residue in the RNA chain and is catalyzed by pseudouridine synthases. The unusual reaction mechanism has become the object of renewed research effort, frequently involving replacement of the substrate uridines with 5-fluorouracil (f(5)U). f(5)U is known to be a potent inhibitor of pseudouridine synthase activity, but the effect varies among the target pseudouridine synthases. Derivatives of f(5)U have previously been detected, which are thought to be either hydrolysis products of covalent enzyme-RNA adducts, or isomerization intermediates. Here we describe the interaction of pseudouridine synthase 1 (Pus1p) with f(5)U-containing tRNA. The interaction described is specific to Pus1p and position 27 in the tRNA anticodon stem, but the enzyme neither forms a covalent adduct nor stalls at a previously identified reaction intermediate of f(5)U. The f(5)U27 residue, as analyzed by a DNAzyme-based assay using TLC and mass spectrometry, displayed physicochemical properties unaltered by the reversible interaction with Pus1p. Thus, Pus1p binds an f(5)U-containing substrate, but, in contrast to other pseudouridine synthases, leaves the chemical structure of f(5)U unchanged. The specific, but nonproductive, interaction demonstrated here thus constitutes an intermediate of Pus turnover, stalled by the presence of f(5)U in an early state of catalysis. Observation of the interaction of Pus1p with fluorescence-labeled tRNA by a real-time readout of fluorescence anisotropy and FRET revealed significant structural distortion of f(5)U-tRNA structure in the stalled intermediate state of pseudouridine catalysis.
- Addresses
- Institute of Pharmacy and Molecular Biotechnology, Department of Chemistry, Heidelberg University, 69120 Heidelberg, Germany.
- Autoren
- Martin Hengesbach
- Felix Voigts-Hoffmann
- Benjamin Hofmann
- Mark Helm
- DOI
- 10.1261/rna.1832510
- eISSN
- 1469-9001
- Externe Identifier
- PubMed Identifier: 20106954
- PubMed Central ID: PMC2822925
- Open access
- false
- ISSN
- 1355-8382
- Ausgabe der Veröffentlichung
- 3
- Zeitschrift
- RNA (New York, N.Y.)
- Schlüsselwörter
- Animals
- Humans
- Mice
- Saccharomyces cerevisiae
- Fluorouracil
- Hydro-Lyases
- RNA, Transfer, Leu
- Pseudouridine
- Fluorescence Resonance Energy Transfer
- RNA Processing, Post-Transcriptional
- Sprache
- eng
- Medium
- Print-Electronic
- Online publication date
- 2010
- Paginierung
- 610 - 620
- Datum der Veröffentlichung
- 2010
- Status
- Published
- Datum der Datenerfassung
- 2010
- Titel
- Formation of a stalled early intermediate of pseudouridine synthesis monitored by real-time FRET.
- Sub types
- Research Support, Non-U.S. Gov't
- research-article
- Journal Article
- Ausgabe der Zeitschrift
- 16
Files
http://rnajournal.cshlp.org/content/16/3/610.full.pdf http://www.rnajournal.org/cgi/reprint/16/3/610 https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20106954/pdf/?tool=EBI https://europepmc.org/articles/PMC2822925?pdf=render
Datenquelle: Europe PubMed Central
- Abstract
- Pseudouridine is the most abundant of more than 100 chemically distinct natural ribonucleotide modifications. Its synthesis consists of an isomerization reaction of a uridine residue in the RNA chain and is catalyzed by pseudouridine synthases. The unusual reaction mechanism has become the object of renewed research effort, frequently involving replacement of the substrate uridines with 5-fluorouracil (f(5)U). f(5)U is known to be a potent inhibitor of pseudouridine synthase activity, but the effect varies among the target pseudouridine synthases. Derivatives of f(5)U have previously been detected, which are thought to be either hydrolysis products of covalent enzyme-RNA adducts, or isomerization intermediates. Here we describe the interaction of pseudouridine synthase 1 (Pus1p) with f(5)U-containing tRNA. The interaction described is specific to Pus1p and position 27 in the tRNA anticodon stem, but the enzyme neither forms a covalent adduct nor stalls at a previously identified reaction intermediate of f(5)U. The f(5)U27 residue, as analyzed by a DNAzyme-based assay using TLC and mass spectrometry, displayed physicochemical properties unaltered by the reversible interaction with Pus1p. Thus, Pus1p binds an f(5)U-containing substrate, but, in contrast to other pseudouridine synthases, leaves the chemical structure of f(5)U unchanged. The specific, but nonproductive, interaction demonstrated here thus constitutes an intermediate of Pus turnover, stalled by the presence of f(5)U in an early state of catalysis. Observation of the interaction of Pus1p with fluorescence-labeled tRNA by a real-time readout of fluorescence anisotropy and FRET revealed significant structural distortion of f(5)U-tRNA structure in the stalled intermediate state of pseudouridine catalysis.
- Autoren
- Martin Hengesbach
- Felix Voigts-Hoffmann
- Benjamin Hofmann
- Mark Helm
- Autoren-URL
- https://www.ncbi.nlm.nih.gov/pubmed/20106954
- DOI
- 10.1261/rna.1832510
- eISSN
- 1469-9001
- Externe Identifier
- PubMed Central ID: PMC2822925
- Ausgabe der Veröffentlichung
- 3
- Zeitschrift
- RNA
- Schlüsselwörter
- Animals
- Fluorescence Resonance Energy Transfer
- Fluorouracil
- Humans
- Hydro-Lyases
- Mice
- Pseudouridine
- RNA Processing, Post-Transcriptional
- RNA, Transfer, Leu
- Saccharomyces cerevisiae
- Sprache
- eng
- Country
- United States
- Paginierung
- 610 - 620
- PII
- rna.1832510
- Datum der Veröffentlichung
- 2010
- Status
- Published
- Datum, an dem der Datensatz öffentlich gemacht wurde
- 2010
- Titel
- Formation of a stalled early intermediate of pseudouridine synthesis monitored by real-time FRET.
- Sub types
- Journal Article
- Research Support, Non-U.S. Gov't
- Ausgabe der Zeitschrift
- 16
Datenquelle: PubMed
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