Analysis of bacterial RNase P RNA and protein interaction by a magnetic biosensor technique.
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.
Li
Dan
D
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35037 Marburg, Germany.
Meyer
Martin H F
MH
Willkomm
Dagmar K
DK
Keusgen
Michael
M
Hartmann
Roland K
RK
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*
D029968:Escherichia coli Proteins; D043262:Ribonuclease P