4.5 Article

Analysis of bacterial RNase P RNA and protein interaction by a magnetic biosensor technique

Journal

BIOCHIMIE
Volume 92, Issue 7, Pages 772-778

Publisher

ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
DOI: 10.1016/j.biochi.2010.02.018

Keywords

Bacterial RNase P; RNA-protein interaction; Magnetic detection; Magnetic beads

Funding

  1. Deutsche Forschungsgemeinschaft [HA 1672/14-1/3, GRK 1384]

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A magnetic sensor technique was applied to analyze the interaction of immobilized bacterial RNase P protein and 3'-biotinylated RNase P RNA bound to streptavidin-coated magnetic beads. Our measurements with three types of beads from different suppliers resulted in K-d values of about 1-2 nM (at 4.5 mM Mg2+ and 150 mM NH4+) for Escherichia coli RNase P RNA and protein, consistent with previous analyses using different techniques. We further measured affinity of the E. coli RNase P protein to chimeric RNase P RNA variants, consisting of an E. coli specificity domain and an engineered archaeal catalytic domain. A bacterial-like 1-bp insertion and 2-nt deletion in the helix P2/P3 region largely improved affinity, providing independent evidence that these elements are crucial for interaction of the two RNase P subunits. Moreover, our study documents that the properties of the streptavidin-coated magnetic beads decide on success or failure of the technique. (C) 2010 Elsevier Masson SAS. All rights reserved.

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