Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using electron paramagnetic resonance spectroscopy

Ribonuclease P (RNase P) is a catalytic ribonucleoprotein (RNP) essential f or tRNA biosynthesis. In Escherichia coli, this RNP complex is composed of a catalytic RNA subunit, M1 RNA, and a protein cofactor, C5 protein. Using the sulfhydryl-specific reagent (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrrol ine-3-methyl)methanethiosulfonate (MTSL), we have introduced a nitroxide sp in label individually at six genetically engineered cysteine residues (i.e. , positions 16, 21, 44, 54, 66, and 106) and the native cysteine residue (i .e., position 113) in C5 protein. The spin label covalently attached to any protein is sensitive to structural changes in its microenvironment. Theref ore, we expected that if the spin label introduced at a particular position in C5 protein was present at the RNA-protein interface, the electron param agnetic resonance (EPR) spectrum of the spin label would be altered upon bi nding of the spin-labeled C5 protein to M1 RNA. The EPR spectra observed wi th the various MTSL-modified mutant derivatives of C5 protein indicate that the spin label attached to the protein at positions 16, 44, 54, 66, and 11 3 is immobilized to varying degrees upon addition of M1 RNA but not in the presence of a catalytically inactive, deletion derivative of M1 RNA. In con trast, the spin label attached to position 21 displays an increased mobilit y upon binding to M1 RNA. The results from this EPR spectroscopy-based appr oach together with those from earlier studies identify residues in C5 prote in which are proximal to M1 RNA in the RNase P holoenzyme complex.