DNA-binding properties of the HMG domain of the lymphoid-specific transcriptional regulator LEF-1.

Abstract

Lymphoid enhancer-binding factor 1 (LEF-11 is a pre-B and T lymphocyte-specific nuclear protein that participates in the regulation of the T-cell antigen receptor (TCR) a 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.

Sequence-specific HMG box factors recognize 10-12 base pair minor groove motifs

“…This region coincides with a stretch of basic amino acids located C-terminal to the HMG box proper, which has been shown to contribute to the affinity of binding in several studies (4,40,41), and more recently has been found to increase the bending angle of a DNA motif complexed to the Lef-1 HMG box (10). Our deletion and footprinting data on the Ste11 HMG box indicate that the C-terminal basic region mediates recognition of the A 11 A 12 3' flank sequence.…”

The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures.

“…The known properties of HU, HMG1, and HMG2 are distinct from their sequence-specific counterparts: IHF in bacteria and LEF1 (the clearest example) in mammals. Both IHF and LEF-1 bind and bend DNA at specific recognition sites (Robertson and Nash 1988;Thompson and Landy 1988;Yang and Nash 1989;Giese et al 1991Giese et al ,1992. The binding of LEF-1 or the presence of intrinsically curved DNA can functionally substitute for IHF in the phage ~ intasome reaction (Goodman and Nash 1989; Giese et al 1992;Goodman et al 1992).…”

The RNA polymerase I transcription factor UBF is a sequence-tolerant HMG-box protein that can recognize structured nucleic acids

“…This conservation presumably accounts for the similar properties of various members of the HMG-box protein family. The insensitivity of UBF to major groove modifications coupled with its sensitivity to the minor groove binding drugs distamycin A, chromomycin A3 and actinomycin D suggests that UBF may be a minor groove binding protein as determining factor), TCF1 (T-Cell Factor-1), and LEF-1 (Lymphoid Enhancer Factor) (41,42). The DNA binding specificity of different HMG-box proteins is presumably dictated by variant amino acids displayed on the DNA binding surface(s) (47,48).…”