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

Venkat Gopalan, Henriette Kühne, Roopa Biswas, Huimei Li, Gary W Brudvig, Sidney Altman

Research output: Contribution to journalArticle

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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- Δ3-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.

Original languageEnglish
Pages (from-to)1705-1714
Number of pages10
JournalBiochemistry
Volume38
Issue number6
DOIs
Publication statusPublished - Feb 9 1999

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Protein Interaction Mapping
Ribonuclease P
Electron Spin Resonance Spectroscopy
Escherichia coli
Paramagnetic resonance
Spectrum Analysis
Spin Labels
Spectroscopy
RNA
Proteins
Ribonucleoproteins
Cysteine
Derivatives
Sulfhydryl Reagents
Catalytic RNA
Holoenzymes
Biosynthesis
Transfer RNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mapping RNA - Protein interactions in ribonuclease P from Escherichia coli using electron paramagnetic resonance spectroscopy. / Gopalan, Venkat; Kühne, Henriette; Biswas, Roopa; Li, Huimei; Brudvig, Gary W; Altman, Sidney.

In: Biochemistry, Vol. 38, No. 6, 09.02.1999, p. 1705-1714.

Research output: Contribution to journalArticle

Gopalan, Venkat ; Kühne, Henriette ; Biswas, Roopa ; Li, Huimei ; Brudvig, Gary W ; Altman, Sidney. / Mapping RNA - Protein interactions in ribonuclease P from Escherichia coli using electron paramagnetic resonance spectroscopy. In: Biochemistry. 1999 ; Vol. 38, No. 6. pp. 1705-1714.
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