Evidence of changes in protease sensitivity and subunit exchange rate on DNA binding by C/EBP.
The transcription factor C/EBP uses a bipartite structural motif to bind DNA. Two protein chains dimerize through a set of amphipathic alpha helices termed the leucine zipper. Highly basic polypeptide regions emerge from the zipper to form a linked set of DNA contact surfaces. In the recently proposed a "scissors grip" model, the paired set of basic regions begin DNA contact at a central point and track in opposite directions along the major groove, forming a molecular clamp around DNA. This model predicts that C/EBP must undertake significant changes in protein conformation as it binds and releases DNA. The basic region of ligand-free C/EBP is highly sensitive to protease digestion. Pronounced resistance to proteolysis occurred when C/EBP associated with its specific DNA substrate. Sequencing of discrete proteolytic fragments showed that prominent sites for proteolysis occur at two junction points predicted by the "scissors grip" model. One junction corresponds to the cleft where the basic regions emerge from the leucine zipper. The other corresponds to a localized nonhelical segment that has been hypothesized to contain an N-cap and facilitate the sharp angulation necessary for the basic region to track continuously in the major groove of DNA.
Shuman
J D
JD
Howard Hughes Research Laboratories, Department of Embryology, Baltimore, MD 21210.
Vinson
C R
CR
McKnight
S L
SL
D000595:Amino Acid Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D007930:Leucine; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011487:Protein Conformation; D014157:Transcription Factors / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D010446:Peptide Fragments; D014157:Transcription Factors; D004247:DNA; D010447:Peptide Hydrolases; D014357:Trypsin; D007930:Leucine