Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-Fe

Roopa Biswas, David W. Ledman, Robert O. Fox, Sidney Altman, Venkat Gopalan

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

The protein subunit of Escherichia coli ribonuclease P (which has a cysteine residue at position 113) and its single cysteine-substituted mutant derivatives (S16C/C113S, K54C/C113S and K66C/C113S) have been modified using a sulfhydryl-specific iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide (EPD-Fe). This reaction converts C5 protein, or its single cysteine-substituted mutant derivatives, into chemical nucleases which are capable of cleaving the cognate RNA ligand, M1 RNA, the catalytic RNA subunit of E. coli RNase P, in the presence of ascorbate and hydrogen peroxide. Cleavages in M1 RNA are expected to occur at positions proximal to the site of contact between the modified residue (in C5 protein) and the ribose units in M1 RNA. When EPD-Fe was used to modify residue Cys16 in C5 protein, hydroxyl radical-mediated cleavages occurred predominantly in the P3 helix of M1 RNA present in the reconstituted holoenzyme. C5 Cys54-EDTA-Fe produced cleavages on the 5' strand of the P4 pseudoknot of M1 RNA, while the cleavages promoted by C5 Cys66-EDTA-Fe were in the loop connecting helices P18 and P2 (J18/2) and the loop (J2/4) preceding the 3' strand of the P4 pseudoknot. However, hydroxyl radical-mediated cleavages in M1 RNA were not evident with Cys113-EDTA-Fe, perhaps indicative of Cys113 being distal from the RNA-protein interface in the RNase P holoenzyme. Our directed hydroxyl radical-mediated footprinting experiments indicate that conserved residues in the RNA and protein subunit of the RNase-P holoenzyme are adjacent to each other and provide structural information essential for understanding the assembly of RNase P. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)19-31
Number of pages13
JournalJournal of Molecular Biology
Volume296
Issue number1
DOIs
StatePublished - Feb 11 2000
Externally publishedYes

Fingerprint

Protein Interaction Mapping
Ribonuclease P
Edetic Acid
Disulfides
RNA
Escherichia coli
Holoenzymes
Hydroxyl Radical
Cysteine
Protein Subunits
Proteins
RNA Cleavage
Catalytic RNA
Ribose
Hydrogen Peroxide
Catalytic Domain
Iron
Ligands

Keywords

  • EDTA-Fe
  • EPD-Fe
  • Footprinting
  • RNA-protein interactions
  • RNase P protein subunit

ASJC Scopus subject areas

  • Virology

Cite this

Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-Fe. / Biswas, Roopa; Ledman, David W.; Fox, Robert O.; Altman, Sidney; Gopalan, Venkat.

In: Journal of Molecular Biology, Vol. 296, No. 1, 11.02.2000, p. 19-31.

Research output: Contribution to journalArticle

Biswas, Roopa ; Ledman, David W. ; Fox, Robert O. ; Altman, Sidney ; Gopalan, Venkat. / Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-Fe. In: Journal of Molecular Biology. 2000 ; Vol. 296, No. 1. pp. 19-31.
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