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 language | English (US) |
|---|---|
| Pages (from-to) | 4186-4192 |
| Number of pages | 7 |
| Journal | Biochemistry |
| Volume | 41 |
| Issue number | 13 |
| DOIs | |
| State | Published - Apr 2 2002 |
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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 journal › Article
}
TY - JOUR
T1 - Proteolytic DNA for mapping protein - DNA interactions
AU - Schmidt, Brian D.
AU - Meares, Claude F.
PY - 2002/4/2
Y1 - 2002/4/2
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0037006985&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037006985&partnerID=8YFLogxK
U2 - 10.1021/bi015582r
DO - 10.1021/bi015582r
M3 - Article
C2 - 11914063
AN - SCOPUS:0037006985
VL - 41
SP - 4186
EP - 4192
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 13
ER -