Abstract
Unlike the DNA-binding domains (DBD) of most eukaryotic transcription factors, Escherichia coli LacI family transcription factors are unable to bind to specific target DNA sequences without a cofactor-binding domain. In the present study, we reconstructed a novel DBD designated as PurHG, which binds constitutively to a 16bp purine repressor operator, by fusion of the purine repressor (PurR) DBD (residues 1-57) and the GAL4 dimerization domain (DD, residues 42-148). Binding of PurHG to DNA requires the dimerization and a hinge helix of PurR DBD. When the PurHG was expressed as a fusion protein in a form of a transcription activator (PurAD) or an artificial nuclear receptor (PurAPR or PurAER) responding to ligand, such as RU486 or β-estradiol, it could regulate the expression of the reporter genes in NIH3T3 cells. The prerequisite region of the GAL4 DD for DNA-binding was amino acid residues from 42 to 98 in the form of PurAD, while the amino acid residues from 42 to 75 were sufficient for ligand-dependent regulation in the form of PurAPR. These results suggest that the dimerization function of the progesterone ligand-binding domain could be substituted for region 76-98 of the GAL4 DD. In summary, the fusion of the PurR DBD and the GAL4 DD generates fully active DNA-binding protein, PurHG, in vitro and in vivo, and these results provide the direct evidence of structural predictions that the proximate positioning of PurR hinge helical regions is critical for DNA-binding.
| Original language | English |
|---|---|
| Pages (from-to) | 334-341 |
| Number of pages | 8 |
| Journal | Biochemical and Biophysical Research Communications |
| Volume | 319 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 2004 Jun 25 |
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All Science Journal Classification (ASJC) codes
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology
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Controlled transcriptional regulation in eukaryotes by a novel transcription factor derived from Escherichia coli purine repressor. / Yeon, Eun Hee; Noh, Ju Young; Kim, Jong Min; Lee, Min Young; Yoon, Sarah; Park, Sang Kyu; Choi, Kang Yell; Kim, Kyung Sup.
In: Biochemical and Biophysical Research Communications, Vol. 319, No. 2, 25.06.2004, p. 334-341.Research output: Contribution to journal › Article
TY - JOUR
T1 - Controlled transcriptional regulation in eukaryotes by a novel transcription factor derived from Escherichia coli purine repressor
AU - Yeon, Eun Hee
AU - Noh, Ju Young
AU - Kim, Jong Min
AU - Lee, Min Young
AU - Yoon, Sarah
AU - Park, Sang Kyu
AU - Choi, Kang Yell
AU - Kim, Kyung Sup
PY - 2004/6/25
Y1 - 2004/6/25
N2 - Unlike the DNA-binding domains (DBD) of most eukaryotic transcription factors, Escherichia coli LacI family transcription factors are unable to bind to specific target DNA sequences without a cofactor-binding domain. In the present study, we reconstructed a novel DBD designated as PurHG, which binds constitutively to a 16bp purine repressor operator, by fusion of the purine repressor (PurR) DBD (residues 1-57) and the GAL4 dimerization domain (DD, residues 42-148). Binding of PurHG to DNA requires the dimerization and a hinge helix of PurR DBD. When the PurHG was expressed as a fusion protein in a form of a transcription activator (PurAD) or an artificial nuclear receptor (PurAPR or PurAER) responding to ligand, such as RU486 or β-estradiol, it could regulate the expression of the reporter genes in NIH3T3 cells. The prerequisite region of the GAL4 DD for DNA-binding was amino acid residues from 42 to 98 in the form of PurAD, while the amino acid residues from 42 to 75 were sufficient for ligand-dependent regulation in the form of PurAPR. These results suggest that the dimerization function of the progesterone ligand-binding domain could be substituted for region 76-98 of the GAL4 DD. In summary, the fusion of the PurR DBD and the GAL4 DD generates fully active DNA-binding protein, PurHG, in vitro and in vivo, and these results provide the direct evidence of structural predictions that the proximate positioning of PurR hinge helical regions is critical for DNA-binding.
AB - Unlike the DNA-binding domains (DBD) of most eukaryotic transcription factors, Escherichia coli LacI family transcription factors are unable to bind to specific target DNA sequences without a cofactor-binding domain. In the present study, we reconstructed a novel DBD designated as PurHG, which binds constitutively to a 16bp purine repressor operator, by fusion of the purine repressor (PurR) DBD (residues 1-57) and the GAL4 dimerization domain (DD, residues 42-148). Binding of PurHG to DNA requires the dimerization and a hinge helix of PurR DBD. When the PurHG was expressed as a fusion protein in a form of a transcription activator (PurAD) or an artificial nuclear receptor (PurAPR or PurAER) responding to ligand, such as RU486 or β-estradiol, it could regulate the expression of the reporter genes in NIH3T3 cells. The prerequisite region of the GAL4 DD for DNA-binding was amino acid residues from 42 to 98 in the form of PurAD, while the amino acid residues from 42 to 75 were sufficient for ligand-dependent regulation in the form of PurAPR. These results suggest that the dimerization function of the progesterone ligand-binding domain could be substituted for region 76-98 of the GAL4 DD. In summary, the fusion of the PurR DBD and the GAL4 DD generates fully active DNA-binding protein, PurHG, in vitro and in vivo, and these results provide the direct evidence of structural predictions that the proximate positioning of PurR hinge helical regions is critical for DNA-binding.
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U2 - 10.1016/j.bbrc.2004.05.007
DO - 10.1016/j.bbrc.2004.05.007
M3 - Article
C2 - 15178411
AN - SCOPUS:2642562760
VL - 319
SP - 334
EP - 341
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 2
ER -