Mapping RNA−Protein Interactions in Ribonuclease P from Escherichia coli Using Electron Paramagnetic Resonance Spectroscopy

Abstract

Ribonuclease P (RNase P) is a catalytic ribonucleoprotein (RNP) essential for 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-Delta3-pyrroline-3-methyl)methanethiosulfonate (MTSL), we have introduced a nitroxide spin 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. Therefore, we expected that if the spin label introduced at a particular position in C5 protein was present at the RNA-protein interface, the electron paramagnetic resonance (EPR) spectrum of the spin label would be altered upon binding of the spin-labeled C5 protein to M1 RNA. The EPR spectra observed with 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 113 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 contrast, the spin label attached to position 21 displays an increased mobility upon binding to M1 RNA. The results from this EPR spectroscopy-based approach together with those from earlier studies identify residues in C5 protein which are proximal to M1 RNA in the RNase P holoenzyme complex.

Ionic interactions between PRNA and P protein in Bacillus subtilis RNase P characterized using a magnetocapture-based assay

“…These residues could also be involved in charge-charge interactions in RNase P holoenzyme. A site-directed spin label incorporated at position E. coli K54 (K53, B. subtilis) has decreased mobility, as indicated by EPR spectroscopy, on addition of the E. coli M1 RNA, further implicating its potential involvement in a protein-RNA interaction (Gopalan et al 1999). In summary, the RNR motif and, possibly, the N terminus are the area(s) to probe to localize the ionic interactions between the PRNA and P protein.…”

Molecular Modeling of the Three-dimensional Structure of the Bacterial RNase P Holoenzyme

“…Details of overexpression and purification of mutant derivatives in E. coli are provided elsewhere. 51,52 After purification of the various mutant derivatives of C5 protein, we performed the modification with EPD-Fe as described 25 and verified the molecular masses of the mutant derivatives, both before and after modification, using ESI mass spectrometric facilities at the OSU Campus Chemical Instrumentation Center. Details on acquisition of the CD spectra as well as the RNase P assays are essentially as outlined in our earlier study.…”

Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-fe 1 1Edited by K. Nagai

“…Since Ser16 was only two residues away from Phe18 and because a Ser to Cys residue substitution causes modest alterations in the chemical character, we chose to replace Ser16 with a Cys residue. Based on the results from our recent EPR spectroscopic studies of M1 RNA-C5 protein interactions, positions 54 and 66 were also selected for Cys mutagenesis modi®cation with EPD-Fe (Gopalan et al, 1999). Although the EPR spectroscopy-based approach facilitated identi®cation of cysteine residues, engineered or otherwise, that might be part of the RNA-protein interface in an RNP complex, it did not yield any information on which residues in M1 RNA are involved in these interactions (Gopalan et al, 1999).…”

“…Based on the results from our recent EPR spectroscopic studies of M1 RNA-C5 protein interactions, positions 54 and 66 were also selected for Cys mutagenesis modi®cation with EPD-Fe (Gopalan et al, 1999). Although the EPR spectroscopy-based approach facilitated identi®cation of cysteine residues, engineered or otherwise, that might be part of the RNA-protein interface in an RNP complex, it did not yield any information on which residues in M1 RNA are involved in these interactions (Gopalan et al, 1999). The EPD-Fe footprinting experiments reported here were performed to identify nucleotide positions in M1 RNA which are proximal to speci®c residues (such as 16, 54 and 66) on C5 protein.…”