The DNA binding domain of GAL4 forms a binuclear metal ion complex

Journal Article · Tue Mar 27 00:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00464a019· OSTI ID:6324632

Pan, T; Colemen, J E ^[1]

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1. Yale Univ. School of Medicine, New Haven, CT (USA)

The transcription factor GAL4 from Saccharomyces cerevisiae requires Zn(II) or Cd(II) for specific recognition of the UAS{sub G} sequence. An N-terminal fragment consisting of the first 63 amino acid residues of GAL4 (GAL4(63)) has been obtained by partial tryptic proteolysis of a cloned and overproduced N-terminal domain of 149 residues, GAL(149). The authors show that GAL4(63) contains the minimal GAL4 DNA binding domain. GAL4(63) binds tightly 1-2 mol of Zn(II) or 2 mol of Cd(II). {sup 113}Cd NMR of {sup 113}Cd(II)-substituted GAL4(63) reveals structural identity between the metal binding domains of GAL4(63) and that of the larger precursor GAL4(149). {sup 113}Cd(II) can be substituted for the Zn(II) in GAL4(63), and two {sup 113}Cd NMR signals are observed at 706 and 669 ppm, both suggesting coordination of {sup 113}Cd(II) to three or four -S{sup {minus}} ligands. With the exception of the N-terminal methionine, the only sulfur-containing residues are the six highly conserved cysteines. High-resolution {sup 1}H NMR of Zn(II)-GAL4(63) and Cd(II)-GAL4(63) show the two proteins to have almost identical conformations and to be present as monomers in solutions up to millimolar concentration. This leads us to postulate that GAL4 does not possess a TFIIIA-like Zn-finger but forms a binuclear metal cluster involving all six cysteines in a cloverleaf -like array. GAL4(63) contains about 60% {alpha}-helix, estimated from circular dichroism. Removal of the native Zn(II) causes substantial unfolding of the secondary structure. Unlike GAL4(149), the resultant apoprotein is not induced to refold by readdition of Zn(II) at low concentrations.

• OSTI ID: 6324632
• Journal Information: Biochemistry; (USA), Vol. 29:12; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA
NUCLEAR MAGNETIC RESONANCE
TRANSCRIPTION FACTORS
AMINO ACID SEQUENCE
CADMIUM 113
CADMIUM COMPOUNDS
CATIONS
CROSS-LINKING
PROTONS
SACCHAROMYCES CEREVISIAE
ZINC COMPOUNDS
BARYONS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CADMIUM ISOTOPES
CHARGED PARTICLES
CHEMICAL REACTIONS
ELEMENTARY PARTICLES
EUMYCOTA
EVEN-ODD NUCLEI
FERMIONS
FUNGI
HADRONS
INTERMEDIATE MASS NUCLEI
IONS
ISOMERIC TRANSITION ISOTOPES
ISOTOPES
MAGNETIC RESONANCE
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
NUCLEIC ACIDS
NUCLEONS
NUCLEOPROTEINS
ORGANIC COMPOUNDS
PLANTS
POLYMERIZATION
PROTEINS
RADIOISOTOPES
RESONANCE
SACCHAROMYCES
STABLE ISOTOPES
YEARS LIVING RADIOISOTOPES
YEASTS
550201* - Biochemistry- Tracer Techniques