PURE, BACTERIALLY EXPRESSED DNA-BINDING DOMAINS OF THE FUNCTIONAL ECDYSTEROID RECEPTOR CAPABLE OF INTERACTING SYNERGISTICALLY WITH THE HSP-27 20-HYDROXYECDYSONE RESPONSE ELEMENT - GST-INDUCED DIMERIZATION OF DNA-BINDING DOMAINS ALTERS CHARACTERISTICS OF THEIR INTERACTION WITH DNA

The steroid hormone 20-hydroxyecdysone (20E) plays a key role in the i nduction and modulation of morphogenetic events throughout Drosophila melanogaster development. Two members of the nuclear receptor superfam ily, the product of the EcR (EcR) and of the ultraspiracle genes (Usp) , heterodimerize to form its functional receptor. To study the recepto r-DNA interaction, critical for regulating 20E-dependent gene expressi on, it is necessary to produce large quantities of EcR and Usp DNA-bin ding domains. Toward this end DNA-binding domains of EcR and Usp (EcRD BD and UspDBD, respectively) were cloned and expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST). However, the results of DNA-binding studies obtained with purified GST-DBDs we re found to be questionable because the fused proteins oligomerized in solution due to the presence of GST. Therefore DBDs were released fro m GST-chimeric proteins by thrombin cleavage and then purified by glut athione-Sepharose 4B chromatography and by gel filtration on Superdex 75 HR. The gel mobility-shift experiments showed that UspDBD exhibited higher affinity than EcRDBD toward a 20-hydroxyecdysone response elem ent from the Drosophila hsp 27 gene (hsp 27pal). Furthermore, formatio n of the heterodimeric EcRDBD-UspDBD complex was observed to be synerg istic when equimolar mixture of both DBDs was incubated with hsp 27pal . Surprisingly, GST-EcRDBD bound hsp 27pal with higher affinity than G ST-UspDBD. This difference was accompanied by the impaired ability of the GST-DBDs to interact synergistically with hsp 27pal. This is the f irst report on expression and purification of the soluble DBDs of the functional ecdysteroid receptor with satisfying yields. Furthermore, o ur results add to the recent findings which indicate the need for caut ion in interpreting the activities of GST fusion proteins. (C) 1998 Ac ademic Press.