Controlled transcriptional regulation in eukaryotes by a novel transcription factor derived from Escherichia coli purine repressor.
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 beta-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.
Yeon
Eun-Hee
EH
Department of Biochemistry and Molecular Biology, Institute of Genetic Science, Yonsei University, College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Republic of Korea.
Noh
Ju-Young
JY
Kim
Jong-Min
JM
Lee
Min-Young
MY
Yoon
Sarah
S
Park
Sang-Kyu
SK
Choi
Kang-Yell
KY
Kim
Kyung-Sup
KS
D001483:Base Sequence; D017931:DNA Primers; D019281:Dimerization; D004926:Escherichia coli / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D011687:Purines / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*
D017931:DNA Primers; D011687:Purines; D011994:Recombinant Proteins; D014157:Transcription Factors