Purification of Drosophila melanogaster ultraspiracle protein and analysis of its A/B region-dependent dimerization behavior in vitro.
Two members of the nuclear receptor superfamily, EcR (ecdysteroid receptor protein) and Usp (Ultraspiracle), heterodimerize to form a functional receptor for the steroid hormone 20-hydroxyecdysone and thus enable it to coordinate morphogenetic events during insect metamorphosis. N-terminally His-tagged Usp was overexpressed in E. coli cells as a non-truncated protein and purified to homogeneity in two chromatographic steps. It was demonstrated that the recombinant receptor specifically binds the ecdysone response element of the hsp27 gene promoter (hsp27EcRE). Moreover, a highly synergistically formed heterodimeric complex with the DNA-binding domain of EcR was observed on hsp27EcRE, but not on the native Usp response element from the chorion s15 gene promoter. Recombinant Usp forms homodimers and homotetramers in the absence of DNA, as judged from gel filtration and chemical crosslinking experiments. Truncation of its N-terminal A/B region changes molecular characteristics of Usp, considerably weakening its oligomerization potential under the same experimental conditions. This contrasts with the results obtained previously for the similarly truncated RXR--a vertebrate homolog of Usp.
Rymarczyk
Grzegorz
G
Institute of Organic Chemistry, Biochemistry and Biotechnology, Division of Biochemistry, Wrocław University of Technology, Wybrzeze Wyspiańskiego 27, 50-370 Wrocław, Poland.
Grad
Iwona
I
Rusek
Agnieszka
A
Oświecimska-Rusin
Kamila
K
Niedziela-Majka
Anita
A
Kochman
Marian
M
Ozyhar
Andrzej
A
D000818:Animals; D002021:Buffers; D002823:Chorion / Q000378:metabolism; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D004331:Drosophila melanogaster; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli; D005822:Genetic Vectors; D015151:Immunoblotting; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D015345:Oligonucleotide Probes; D010957:Plasmids / Q000235:genetics; D011987:Receptors, Steroid / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification
D002021:Buffers; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D009363:Neoplasm Proteins; D015345:Oligonucleotide Probes; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D014157:Transcription Factors; C066030:USP protein, Drosophila; C034808:ecdysteroid receptor; D004247:DNA