Assembly of b/HLH/z proteins c-Myc, Max, and Mad1 with cognate DNA: importance of protein-protein and protein-DNA interactions

Abstract

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.

Figure 1

Figure 1A. MLP DNA oligonucleotide (○), LCR DNA oligonucleotide (●) and mLCR DNA oligonucleotide (□) titrated with Max Protein. The concentration of MLP was 20nM, LCR and mLCR were 100nM. Anisotropy change was shown as Y value by increasing the Max protein concentration (X value). The temperature was 22 °C. The titration curves indicate stronger binding affinity for MLP than LCR. Figure 1B. Normalized binding of Max protein to MLP (○) and LCR (●) DNA oligonucleotides. The normalized values represent the fraction bound. Experimental conditions are as described for Figure 1A.

Figure 1

Figure 1A. MLP DNA oligonucleotide (○), LCR DNA oligonucleotide (●) and mLCR DNA oligonucleotide (□) titrated with Max Protein. The concentration of MLP was 20nM, LCR and mLCR were 100nM. Anisotropy change was shown as Y value by increasing the Max protein concentration (X value). The temperature was 22 °C. The titration curves indicate stronger binding affinity for MLP than LCR. Figure 1B. Normalized binding of Max protein to MLP (○) and LCR (●) DNA oligonucleotides. The normalized values represent the fraction bound. Experimental conditions are as described for Figure 1A.

Figure 2

Anisotropy change for the interaction between Max protein and 3 different oligonucleotides. The concentration of Ebox (◇), half-Ebox (□) and non-Ebox (○) oligonucleotides were 50 nM. Temperature was 21 °C. The base sequences for the oligonucleotides are given in Material and Methods. The equilibrium constants obtained from the anisotropy change were listed in Table 2.

Figure 3

Anisotropy change for the interaction between Myc-Max protein and Ebox (□) and half-Ebox (△) oligonucleotides. The experimental conditions are the same as for Figure 2.

Figure 4

Anisotropy change for the interaction between Mad-Max protein and Ebox (□) and half-Ebox (△) oligonucleotides. The experimental conditions are the same as for Figure 2.

Figure 5

Dimerization of Max-Max (◇), Myc-Max (□), and Mad-Max (○). Max was labeled with TRITC. Labeled Max (100 nM) was titrated with increasing concentrations of unlabeled Max, Myc or Mad. For these experiments, GST was enzymatically removed from Myc. The temperature was 20 °C. The larger anisotropy change for Mad is attributed to the larger molecular mass.

Figure 6

Schematic diagram of monomer, dimer and DNA interactions.

Figure 7

Titration of Max-DNA (50 nM) with increasing concentrations of Myc protein. The E-box oligonucleotide labeled with 5′ FITC was used for the titration. Temperature was 21 °C.