Recognition of DNA by GAL4 in solution: use of a monomeric protein-DNA complex for study by NMR.
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)
Baleja
J D
JD
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Mau
T
T
Wagner
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008961:Models, Structural; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA