Characterization of the DNA Binding Properties ofInterleukin Enhancer Binding Factor
| Autor: | Chien-Hui Sun, 孫千惠 |
|---|---|
| Rok vydání: | 2003 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 91 Interleukin enhancer binding factor (ILF) is a transcription factor which consists of 655 amino acids and binds to the IL-2 promoter. Although the function of ILF is not well understood, previous studies suggest that they might have a positive role in regulating IL-2 gene expression. The DNA-binding domain of ILF is a member of the winged helix/ forkhead family and consists of four α-helices, three β-strands, one type I turn, and one wing, arranged in the order H1-T1-S1-H2-H3-S2-W1-S3-H4. Interestingly, in contrast to other proteins of this family, the DNA-binding domain of ILF contains a C-terminal -helix in place of a typical Wing 2. This extra α-helix is stabilized by the hydrophobic interactions between H4 and H1. This structural difference may be responsible for the different DNA-binding specificity of ILF compared to other winged helix /forkhead proteins. Ets-1, a member of winged helix transcription factors, also has the Helix 4 structure and has been shown to exhibit an intramolecular autoinhibition mechanism to regulate its binding to DNA. Additional two α-helices (HI-1 and HI-2) at the N-terminus of the DNA-binding domain of ETS-1 interact with H4 to form an inhibitory module to repress its DNA-binding affinity. In addition, the calcium-dependent phosphorylation represses the DNA binding of Ets-1 by reinforcing autoinhibition. Four serine residues mediated this inhibitory effect are located at the N-terminus of the DNA-binding domain. Based on secondary structure analysis, we found that the N terminal region of the DNA-binding domain of ILF may form two α-helices. Therefore, we propose that ILF may regulate the DNA binding activity by autoinhibition mechanism that was used by Ets-1. In this study we expressed four fragments of ILF101(251-351), ILF131(221-351), ILF141(211-351), and ILF153(202-354) in E. coli and study the relative binding affinity of four fragments of ILF with different DNA probes by electrophoretic mobility shift assay (EMSA). The EMSA study showed that the relative binding affinities of the four proteins with the probe CCCGGCAAAACATCAA were ILF153, ILF101 > ILF131, ILF141. The Kd values of the fragments of ILF and the probes are ranging from 90-420 nM. We also found that the probes with the sequences GTAAACA or ATAAACA can specifically bind to the DNA-binding domain of ILF. The study on the phosphorylation effect of the N-terminus of the DNA-binding domain of ILF on its DNA binding is ongoing. This study demonstrates a similar autoinhibition mechanism among the winged helix transcription factors. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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