DNA-binding properties of the HMG domain of the lymphoid-specific transcriptional regulator LEF-1.
Lymphoid enhancer-binding factor 1 (LEF-1) is a pre-B and T lymphocyte-specific nuclear protein that participates in the regulation of the T-cell antigen receptor (TCR) alpha enhancer by binding to the nucleotide sequence 5'-CCTTTGAA. LEF-1 protein shares with the nonhistone high mobility group protein 1 (HMG-1) and several transcriptional regulators a single region of amino acid homology, termed the HMG box, which has been implicated in DNA binding. Here, we report the biochemical analysis of the interaction of this novel structural motif with DNA. First, amino- or carboxy-terminal truncations of the LEF-1 polypeptide delineated the HMG box as the DNA-binding domain. We purified to homogeneity a LEF-HMG domain peptide expressed in Escherichia coli and determined the equilibrium constant for specific binding to DNA as 1 x 10(-9) M. Second, cotranslation of wild-type and various truncated LEF-1 polypeptides did not generate any DNA-binding heterodimers, suggesting that LEF-1 can bind DNA as a monomer. Third, methylation interference analysis indicated that the HMG domain specifically contacts DNA on one side of the double helix. Finally, changes of amino acids that are conserved among various members of the family of HMG-box proteins decreased the affinity of DNA binding by one to three orders of magnitude. Together, these data define the characteristics of specific DNA-binding by the HMG domain of LEF-1.
Giese
K
K
Howard Hughes Medical Institute, Department of Microbiology, University of California, San Francisco 94143-0414.
Amsterdam
A
A
Grosschedl
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D006609:High Mobility Group Proteins / Q000235:genetics*; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry*; D051821:Lymphoid Enhancer-Binding Factor 1; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D009690:Nucleic Acid Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; C497573:LEF1 protein, human; D051821:Lymphoid Enhancer-Binding Factor 1; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors