title: Analysis of bacterial RNase P RNA and protein interaction by a magnetic biosensor technique.
abstract: 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 Mg(2+) and 150 mM NH(4)(+)) 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.
authors.affiliation: Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35037 Marburg, Germany.
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: Martin H F
authors.initials: MH
authors.lastname: Meyer
authors.forename: Dagmar K
authors.initials: DK
authors.lastname: Willkomm
authors.forename: Michael
authors.initials: M
authors.lastname: Keusgen
authors.forename: Roland K
authors.initials: RK
authors.lastname: Hartmann
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008280:Magnetics; D008863:Microspheres; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D043262:Ribonuclease P / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D043262:Ribonuclease P