Regensburg 2004 – wissenschaftliches Programm

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SYLS: Life Sciences on the Nanometer Scale - Physics Meets Biology

SYLS 3: Symposium "Life Sciences on the Nanometer Scale - Physics Meets Biology"

SYLS 3.12: Poster

Mittwoch, 10. März 2004, 16:00–18:30, B

A fluorescence anisotropy-based activity assay for the RNA-Helicase DbpA — •Niklas Nachtmann and Dagmar Klostermeier — Experimental Physics IV, University of Bayreuth, Bayreuth

The Escherichia coli protein DbpA is an RNA-helicase involved in ribosome biogenesis. It comprises conserved helicase motifs, such as a Walker A motif and a DEAD box involved in ATP binding and hydrolysis, an arginine-rich motif that mediates RNA binding, and a SAT motif responsible for coupling of ATPase and RNA unwinding activities.

We have developed a DbpA helicase activity assay using a fluorescein-labeled model substrate and fluorescence anisotropy. The fluorescently labeled substrate binds to DbpA with high affinity, and the unwinding of its short double-stranded region can be followed via a concomitant decrease in fluorescein anisotropy. This anisotropy decrease is only observed in the presence of ATP, not ADP, consistent with ATP-dependent helix unwinding. Furthermore, a mutant of DbpA in which the conserved SAT motif has been converted to AAA does not exhibit helicase activity towards this substrate, as monitored by fluorescence anisotropy.

Despite minor sequence differences in the corresponding ribosomal RNA region, this activity test is applicable to the homologous RNA helicase YxiN from Bacillus subtilis. This anisotropy-based activity test will be an invaluable means to assay helicase constructs manipulated for single molecule FRET experiments for wild-type like activity.