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

Spenkuch F, Hinze G, Kellner S, Kreutz C, Micura R, Basché T, Helm M - Nucleic Acids Res. (2014)

Bottom Line: 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 Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55128 Mainz, Germany.

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Results of MST experiments. (a) Typical set of thermophoresis curves for protein binding. (b) Titration curves for tmTruB binding (filled symbols) fitted to a one-to-one binding model (solid lines). Empty symbols represent the same samples after addition of unlabeled U55-tRNA in excess (chase). Note that negative values in the chase experiments are presumably caused by the different spectral properties of the bound and the unbound species. Error bars are standard deviations from triplicate measurements. (c) Typical thermophoresis curves in chase experiments by addition of unlabeled U55-tRNA.
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Figure 6: Results of MST experiments. (a) Typical set of thermophoresis curves for protein binding. (b) Titration curves for tmTruB binding (filled symbols) fitted to a one-to-one binding model (solid lines). Empty symbols represent the same samples after addition of unlabeled U55-tRNA in excess (chase). Note that negative values in the chase experiments are presumably caused by the different spectral properties of the bound and the unbound species. Error bars are standard deviations from triplicate measurements. (c) Typical thermophoresis curves in chase experiments by addition of unlabeled U55-tRNA.

Mentions: As a further method for independent verification, we chose MST, an emerging method that has only scarcely been applied to RNA-protein interactions until now, but is highly suitable for working with fluorescently labeled macromolecules (52). Figure 6a shows the primary data of a typical titration experiment using C49-U55-tRNA (see Supplementary Figure S4 for curves of all constructs). Application of MST to the tRNA-TruB interaction (ctRNA = 50 nM) produced highly reproducible binding curves (Figure 6b) and corresponding Kd values in a remarkably short turnaround time. Akin to the fluorescence measurements above, MST measurements are conducted under buffer conditions considered native. In addition, we found that MST facilitates an assessment of the homogeneity of the protein preparation (Supplementary Figure S5) and reproduces Kd values after 1–3 month intervals with a factor of 2 as maximum deviation (Supplementary Figure S6). Two sets of similar binding curves are apparent, with both U33-tRNAs corresponding to higher affinity (filled squares and circles in Figure 6b) than the C49 derivatives (filled triangles and diamonds).


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

Spenkuch F, Hinze G, Kellner S, Kreutz C, Micura R, Basché T, Helm M - Nucleic Acids Res. (2014)

Results of MST experiments. (a) Typical set of thermophoresis curves for protein binding. (b) Titration curves for tmTruB binding (filled symbols) fitted to a one-to-one binding model (solid lines). Empty symbols represent the same samples after addition of unlabeled U55-tRNA in excess (chase). Note that negative values in the chase experiments are presumably caused by the different spectral properties of the bound and the unbound species. Error bars are standard deviations from triplicate measurements. (c) Typical thermophoresis curves in chase experiments by addition of unlabeled U55-tRNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4227767&req=5

Figure 6: Results of MST experiments. (a) Typical set of thermophoresis curves for protein binding. (b) Titration curves for tmTruB binding (filled symbols) fitted to a one-to-one binding model (solid lines). Empty symbols represent the same samples after addition of unlabeled U55-tRNA in excess (chase). Note that negative values in the chase experiments are presumably caused by the different spectral properties of the bound and the unbound species. Error bars are standard deviations from triplicate measurements. (c) Typical thermophoresis curves in chase experiments by addition of unlabeled U55-tRNA.
Mentions: As a further method for independent verification, we chose MST, an emerging method that has only scarcely been applied to RNA-protein interactions until now, but is highly suitable for working with fluorescently labeled macromolecules (52). Figure 6a shows the primary data of a typical titration experiment using C49-U55-tRNA (see Supplementary Figure S4 for curves of all constructs). Application of MST to the tRNA-TruB interaction (ctRNA = 50 nM) produced highly reproducible binding curves (Figure 6b) and corresponding Kd values in a remarkably short turnaround time. Akin to the fluorescence measurements above, MST measurements are conducted under buffer conditions considered native. In addition, we found that MST facilitates an assessment of the homogeneity of the protein preparation (Supplementary Figure S5) and reproduces Kd values after 1–3 month intervals with a factor of 2 as maximum deviation (Supplementary Figure S6). Two sets of similar binding curves are apparent, with both U33-tRNAs corresponding to higher affinity (filled squares and circles in Figure 6b) than the C49 derivatives (filled triangles and diamonds).

Bottom Line: 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 Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55128 Mainz, Germany.

Show MeSH
Related in: MedlinePlus