Expression and function of a human thyroid hormone receptor-derived DNA-binding domain protein.
DNA binding domain proteins (DBDP) were prepared using a pET construct containing an insert coding for amino acids 49-122 of human thyroid hormone receptor (hTR) alpha and 103-179 of hTR beta. These proteins were expressed in Escherichia coli strain BL21 (DE3)-plysS after induction by isopropyl-D-thiogalactopyranoside (IPTG). The hTR alpha and hTR beta DBDP contain respectively 79 and 82 amino acids, including an amino terminal 4 amino acid extension derived from pET-3a or the synthesized initiation codon. Using a gel shift assay, both DBDPs were found to bind to a DNA oligonucleotide containing a thyroid hormone response element (TRE). The DBDPs competed with full length hTR alpha 1 for binding to the oligonucleotide. Apo-DBDPs (Zn2+ released by low pH) failed to bind to the palindromic TRE. DNA binding is restored however if apo-DBDP is preincubated in 500 microM Zn2+. When the DBDPs were expressed in COS-7 cells using a pCB6+ expression vector, they did not induce expression of a TRE-CAT fusion gene. hTR DBDPs thus can bind to DNA, presumably as monomers, since they do not contain the leucine zipper-like motif for dimerization. In COS-7 cells, they fail to cause transactivation of a TRE-CAT fusion gene. It is inferred that this may be because the DBDPs are not translocated to the nucleus or lack a transactivation domain.
Hu
R M
RM
Department of Medicine, University of Chicago, IL 60637.
Sakurai
A
A
Miyamoto
T
T
DeGroot
L J
LJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011988:Receptors, Thyroid Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011988:Receptors, Thyroid Hormone; D007544:Isopropyl Thiogalactoside; D015500:Chloramphenicol O-Acetyltransferase