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Default Recognition of DNA by GAL4 in solution: use of a monomeric protein-DNA complex for st

Recognition of DNA by GAL4 in solution: use of a monomeric protein-DNA complex for study by NMR.

Related Articles Recognition of DNA by GAL4 in solution: use of a monomeric protein-DNA complex for study by NMR.

Biochemistry. 1994 Mar 15;33(10):3071-8

Authors: Baleja JD, Mau T, Wagner G

The complex of a monomer of GAL4 with DNA has been investigated by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Previous X-ray analysis has revealed a structure in which a dimer of the N-terminal 65-residue fragment of GAL4 forms a complex, 27 kDa in molecular mass, with a 19 base pair full-binding-site DNA [Marmorstein, R., Carey, M., Ptashne, M., & Harrison, S. C. (1992) Nature 356, 408-414]. We have developed a smaller system, half in molecular mass, which is amenable for detailed analysis using NMR. Titration of a 10 base pair half-binding-site DNA with GAL4-(65) shows 1:1 binding, illustrating that one monomer of the protein binds in a specific manner to half-site DNA. The components of the protein-DNA complex are mainly in fast exchange on the NMR chemical shift time scale, with an equilibrium dissociation constant of 161 +/- 12 microM. With a basis of chemical shift data for free GAL4 protein and for the free half-site DNA, the fast exchange facilitates 1H resonance assignments in the complex since cross-peak positions can be examined at different protein:DNA ratios. Chemical shift changes in the DNA reveal the base pairs that are important for recognition by GAL4. Intermolecular NOE cross-peaks are also observed in spectra of the protein-DNA complex. Their identification places the C-terminal end of the first alpha-helix (residues 12-17) in a position such that the amino acids are able to read the DNA sequence in a manner entirely consistent with the X-ray structure of the related complex.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 8130221 [PubMed - indexed for MEDLINE]



Source: PubMed
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