Interaction of the myogenic determination factor myogenin with E12 and a DNA target: mechanism and kinetics.
The myogenic determination factors MyoD, myogenin, myf5, and MRF4 are members of the basic helix-loop-helix (bHLH) family of transcription factors and crucial agents of myogenesis. The bHLH regions of these proteins enable them to dimerize with E proteins, another class of the bHLH family, and to bind a specific DNA element known as an E box (CANNTG consensus sequence), which results in the activation of muscle-specific gene expression. As a model for such assembly of the myogenic determination factor/E protein-DNA ternary complex, we have studied the physiologically relevant association of myogenin, E12, and the 3' E box of the acetylcholine receptor (AChR) alpha-subunit gene enhancer. Using the technique of electrophoretic mobility shift assay combined with order-of-addition and time-course experiments, we find that heterodimerization of myogenin with E12 occurs prior to DNA-binding. In addition, we deduce the dissociation (Kd) and rate (k) constants for each step in the formation of the myogenin/E12-DNA ternary complex. Kinetic simulations indicate that at 37 degrees C myogenin and E12 heterodimerize with a Kd of 36 microM (k(on) of 573 M(-1) x s(-1) and k(off )of 0.0205 x s(-1)), and that subsequently the heterodimer binds the AChR alpha-subunit gene enhancer 3' E box with a Kd of 8.8 nM (with possible k(on) and k(off) values ranging from 1.0x10(8) to 14.1x10(8) M(-1) x s(-1), and 0.875 to 12.3 s(-1), respectively).
Spinner
Daryl S
DS
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook 11794-5215, USA.
Liu
Shaohua
S
Wang
Shao-Wen
SW
Schmidt
Jakob
J
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004742:Enhancer Elements, Genetic / Q000235:genetics; D018257:Helix-Loop-Helix Motifs; D007700:Kinetics; D008969:Molecular Sequence Data; D018008:Myogenin / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits; D011978:Receptors, Nicotinic / Q000235:genetics; D020218:Response Elements / Q000235:genetics*; D051820:TCF Transcription Factors; D013816:Thermodynamics; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors
D002843:Chromatin; D004268:DNA-Binding Proteins; D018008:Myogenin; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D004247:DNA