Kinetic studies of B/HLH/Z transcription factors Myc/Max/Mad

Myc, Mad and Max proteins belong to the basic helix-loop-helix leucine zipper family of transcription factors. They bind to a specific hexanucleotide element of DNA, the E-box (CACGTG). In order to be biologically active, Myc and Mad require dimerization with Max. For the route of complex assembly of these dimers, there are two proposed pathways. Monomer pathway: Two monomers bind DNA sequentially and assemble their dimerization interface while bound to DNA. Dimer pathway: Two monomers form a dimer first, and then associate with DNA.;Stopped flow polarization was utilized to determine the rate of the individual steps in the assembly pathway. Individual rate constants were assigned to dimer formation, DNA binding of dimeric Max-Max, Myc-Max and Mad-Max and DNA binding of monomeric transcription factors. The kinetic effects of Myc-Max, Mad-Max and Max-Max dimerization and the temperature dependence of these reactions were measured and compared. Stopped-flow anisotropy data showed that Myc-Max dimerization had ∼5-fold and ∼2-fold higher rate constant than Max-Max and Mad-Max, respectively. The Myc, Max and Mad dimerization rates were found to be concentration independent suggesting conformational changes were rate limiting. There was no significant difference in the Arrhenius activation energies calculated for the dimerization of Myc, Max and Mad.;The rate constants for the DNA binding of Max-Max homodimer, Myc-Max and Mad-Max heterodimers were also studied as a function of temperature and concentration. Results showed that the average rate constants for the Max-Max-DNA and Myc-Max-DNA are 1.7 and 1.5 fold higher than for the Mad-Max-DNA, respectively. Concentration dependence revealed that the interactions between the protein dimers Max-Max, Myc-Max, Mad-Max and the E-box DNA are concentration independent.;To compare the monomer and dimer pathways, the DNA binding rates of monomeric Max and Myc were studied. Results showed that both monomeric and dimeric transcription factors recognize the palindromic E-Box at similar rates. Since the rate of dimer formation is slower than the DNA binding rates, the monomer pathway was kinetically favored over the dimer pathway.