Giese K, Amsterdam A, Grosschedl R
Howard Hughes Medical Institute, Department of Microbiology, University of California, San Francisco 94143-0414.
Genes Dev. 1991 Dec;5(12B):2567-78. doi: 10.1101/gad.5.12b.2567.
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.
淋巴样增强子结合因子1（LEF-1）是一种前B细胞和T淋巴细胞特异性核蛋白，它通过与核苷酸序列5'-CCTTTGAA结合来参与T细胞抗原受体（TCR）α增强子的调控。LEF-1蛋白与非组蛋白高迁移率族蛋白1（HMG-1）以及几种转录调节因子共享一个单一的氨基酸同源区域，称为HMG框，该区域与DNA结合有关。在此，我们报告了这种新型结构基序与DNA相互作用的生化分析。首先，LEF-1多肽的氨基或羧基末端截短将HMG框确定为DNA结合结构域。我们将在大肠杆菌中表达的LEF-HMG结构域肽纯化至同质，并确定其与DNA特异性结合的平衡常数为1×10⁻⁹ M。其次，野生型和各种截短的LEF-1多肽的共翻译未产生任何DNA结合异二聚体，这表明LEF-1可以作为单体结合DNA。第三，甲基化干扰分析表明，HMG结构域在双螺旋的一侧特异性接触DNA。最后，HMG框蛋白家族各成员中保守的氨基酸变化使DNA结合亲和力降低了一到三个数量级。总之，这些数据定义了LEF-1的HMG结构域特异性DNA结合的特征。
