Kinetic analysis of the interaction of b/HLH/Z transcription factors Myc, Max, and Mad with cognate DNA.
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). 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. In the monomer pathway, two monomers bind DNA sequentially and assemble their dimerization interface while bound to DNA. In the dimer pathway, two monomers form a dimer first prior to association with DNA. The monomer pathway is kinetically favored. In this report, stopped-flow polarization was utilized to determine the rates and temperature dependence of all of the individual steps for both assembly pathways. Myc.Max dimerization had a rate constant approximately 5- and approximately 2-fold higher than those of Max.Max and Mad.Max dimerization, respectively. The protein dimerization rates as well as the dimer-DNA rates were found to be independent of concentration, suggesting conformational changes were rate-limiting. The Arrhenius activation energies for the dimerization of Myc, Mad, and Max with Max were 20.4 +/- 0.8, 29 +/- 0.6, and 40 +/- 0.2 kJ/mol, respectively. Further, rate constants for Max.Max homodimer DNA binding are significantly higher than for Myc.Max and Mad.Max heterodimers binding to DNA. Monomer-DNA binding showed a faster rate than dimer-DNA binding. These studies show the rate-limiting step for the dimer pathway is the formation of protein dimers, and this reaction is slower than formation of protein dimers on the DNA interface, kinetically favoring the monomer pathway.
Ecevit
Ozgur
O
Department of Chemistry and Biochemistry, Hunter College and Graduate Center of the City University of New York, New York, New York 10065, USA.
Khan
Mateen A
MA
Goss
Dixie J
DJ
D000595:Amino Acid Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry*; D004247:DNA / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D051792:Basic Helix-Loop-Helix Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D004247:DNA; D016271:Proto-Oncogene Proteins c-myc; D014157:Transcription Factors; C067850:Myc associated factor X