Expression, purification, and functional analysis of the DNA binding domain of the nuclear receptor Rev-erb beta.
Rev-erb beta is a member of the nuclear receptor superfamily, which includes a group of transcription factors involved in the response to steroids, vitamin D, retinoic acids, and other lipophilic molecules. The Rev-erb nuclear receptors exist at least in two forms, namely alpha and beta, with a high degree of evolutionary conservation at the level of the DNA binding domain. Nevertheless, the exact type of DNA binding of these proteins is not fully understood. In order to get insight into this DNA binding mechanism we obtained a pure and functional homogeneous recombinant protein in bacteria corresponding to the DNA binding domain of Rev-erb beta (REDBD). REDBD interacts with oligonucleotides containing an A/T-rich sequence preceding a single AGG-TCA site (Rev-RE) or with an AGGTCA direct repeat separated by 2 bp (Rev-DR2). These results, and the affinity parameters of the interaction between REDBD and Rev-RE or Rev-DR2, indicate that our REDBD preparation is capable of specific and tight binding to DNA targets.
Terenzi
H
H
Institut Pasteur, Unité d'Expression des Gènes Eucaryotes, Paris, France. hterenzi@pasteur.fr
Cassia
R O
RO
Zakin
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D004268:DNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D004247:DNA