Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor

 

Felix Spenkuch, Gerald Hinze, Stefanie Kellner, Christoph Kreutz, Ronald Micura, Thomas Basché & Mark Helm

Nucleic Acids Research
2014 vol: 42(20) pp: 12735–12745 doi: 10.1093/nar/gku908

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.

View Publication

Topics: Competition assay, Enzymes, Nucleic acids, RNA, Monolith – MicroScale Thermophoresis, MST, Proteins, Publications

 

 

Previous Article
Rational design of a bisphenol A aptamer selective surface-enhanced Raman scattering nanoprobe
Rational design of a bisphenol A aptamer selective surface-enhanced Raman scattering nanoprobe

Up next
Myosin VI must dimerize and deploy its unusual lever arm in order to perform its cellular roles
Myosin VI must dimerize and deploy its unusual lever arm in order to perform its cellular roles

×

Sign up to receive
the latest NanoTemper news, product updates, event announcements and more

First Name
Last Name
Company Name
State
Province
State
Region
State
Canton
Agree to Privacy Policy*
*I have fully read, understood and agree to the Privacy Policy. I accept the storing and processing of my personal data by NanoTemper as described in the Privacy Policy.

By completing this form, you provide us consent to contact you with educational content, news and information about our products, services and events. You may unsubscribe at any time.
Thank you!
Error - something went wrong!