Assembly of b/HLH/z proteins c-Myc, Max, and Mad1 with cognate DNA: importance of protein-protein and protein-DNA interactions.
Among the best characterized of the transcription factors are the b/HLH/z proteins: USF, Max, Myc, and Mad. These proteins bind to the DNA E-box, a six base pair sequence, CACGTG. Max and Myc form a heterodimer that has strong oncogenic potential but can also repress transcription, while Mad and Max form a heterodimer that acts as a transcription repressor. We have used fluorescence anisotropy to measure protein-protein and protein-DNA affinity. The specific binding between MLP DNA and Max (K = 2.2 +/- 0.5 nM) is about 10-fold higher affinity than LCR DNA and about 100-fold higher than for a nonspecific DNA. USF has a similar binding affinity as Max to MLP DNA (K = 15 +/- 10 nM), but Max binds more tightly to LCR and nonspecific DNA. A series of oligonucleotides designated E-box, half-E-box, and non-E-box were constructed to examine the effects of DNA sequence. The binding results indicate that for Max protein most of the binding energy can be attributed to individual elements with little cooperativity among the two halves of the E-box. Further studies measured the equilibria for the entire thermodynamic cycle of monomer-dimer-DNA interactions. Surprisingly, the affinity of the Max monomer-DNA for the second monomer was greatly reduced (K for the first monomer in the nanomolar range and for the second monomer in the micromolar range). Looked at from the perspective of the Max protein, the binding of DNA to Max significantly reduces the affinity of the Max protein for the second monomer, whether the second monomer is Myc, Mad, or Max. These data suggest the importance of protein-protein interactions in assembly of a transcription initiation complex.
Hu
Jianzhong
J
Department of Chemistry, Hunter College, and Graduate Center of the City University of New York, New York, New York 10021, USA.
Banerjee
Anamika
A
Goss
Dixie J
DJ
D016880:Anisotropy; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D019281:Dimerization; D018257:Helix-Loop-Helix Motifs; D016350:Leucine Zippers; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D013816:Thermodynamics; D014157:Transcription Factors / Q000378:metabolism*
D050976:Basic-Leucine Zipper Transcription Factors; C067850:Myc associated factor X; D016271:Proto-Oncogene Proteins c-myc; D014157:Transcription Factors; D004247:DNA