Proteolytic DNA for mapping protein - DNA interactions

Brian D. Schmidt, Claude F. Meares

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

We describe a technique to determine sites on proteins involved in protein-DNA interactions. DNA was synthesized via polymerase chain reaction (PCR) to produce four polynucleotide products with phosphorothioate nucleotides at the A, T, G, or C residues. Limited conjugation with the chemical protease FeBABE results in the surface of DNA being randomly labeled at the phosphorothioate sites with this protein-cleaving reagent. After formation of a protein-DNA complex, the proteolytic DNA can be activated to cleave the protein backbone at sites near the DNA. This technique was used to study the bacterial RNA polymerase/lacUV5 DNA open promoter complex, about which significant structural information is available. Cleavage sites on the two largest subunits of RNA polymerase, β and β′, agree well with a recent model based on the crystal structure of the core enzyme α2ββ′ [Naryshkin, N., Revyakin, A., Kim, Y., Mekler, V., and Ebright, R. H. (2000) Cell 101,601-611]. The cleavage site present on α supports previous studies regarding DNA binding regions of the α subunit. Cleavage sites identified throughout the σ70 subunit help to orient it with respect to the open promoter complex.

Original languageEnglish (US)
Pages (from-to)4186-4192
Number of pages7
JournalBiochemistry
Volume41
Issue number13
DOIs
StatePublished - Apr 2 2002

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Protein Interaction Mapping
DNA
Proteins
DNA-Directed RNA Polymerases
Bacterial RNA
Polynucleotides
Polymerase chain reaction
Peptide Hydrolases
Nucleotides
Crystal structure

ASJC Scopus subject areas

  • Biochemistry

Cite this

Proteolytic DNA for mapping protein - DNA interactions. / Schmidt, Brian D.; Meares, Claude F.

In: Biochemistry, Vol. 41, No. 13, 02.04.2002, p. 4186-4192.

Research output: Contribution to journalArticle

Schmidt, BD & Meares, CF 2002, 'Proteolytic DNA for mapping protein - DNA interactions', Biochemistry, vol. 41, no. 13, pp. 4186-4192. https://doi.org/10.1021/bi015582r
Schmidt, Brian D. ; Meares, Claude F. / Proteolytic DNA for mapping protein - DNA interactions. In: Biochemistry. 2002 ; Vol. 41, No. 13. pp. 4186-4192.
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