Diallo Ericka M, Thompson Deborah L, Koenig Ronald J
Division of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109-0678, USA.
Protein Expr Purif. 2005 Apr;40(2):292-8. doi: 10.1016/j.pep.2005.01.007.
Thyroid hormone receptors (TRs) are ligand-activated transcription factors that mediate the biological effects of thyroid hormone (T3) by binding to thyroid hormone response elements (TREs), typically located in the promoter regions of T3-responsive genes. It is generally held that T3-induced gene activation is mediated by retinoid X receptor (RXR)-TR heterodimers. Although TR homodimers can bind to some TREs, T3 destabilizes this interaction, calling into question the ability of TR to activate transcription in the absence of RXR. TR-DNA binding has been difficult to study in vitro because mammalian TRs are notoriously difficult to produce in Escherichia coli. We considered that this may be due to codon bias. Therefore, we produced TRbeta1 in E. coli Rosetta 2(DE3) which contains a plasmid that overexpresses the tRNAs corresponding to the seven rarest E. coli codons. This resulted in an improved yield of full-length TRbeta1, which we then used in electrophoretic mobility shift assays. We found the coactivator TIF2 greatly enhances binding of T3-occupied TRs to a subset of TREs, suggesting TRs may activate transcription from these TREs in an RXR-independent manner.
甲状腺激素受体（TRs）是配体激活的转录因子，通过与通常位于T3反应基因启动子区域的甲状腺激素反应元件（TREs）结合来介导甲状腺激素（T3）的生物学效应。一般认为，T3诱导的基因激活是由视黄酸X受体（RXR）-TR异二聚体介导的。虽然TR同二聚体可以与一些TREs结合，但T3会破坏这种相互作用，这使得在没有RXR的情况下TR激活转录的能力受到质疑。由于哺乳动物TRs在大肠杆菌中 notoriously difficult to produce，因此在体外研究TR-DNA结合一直很困难。我们认为这可能是由于密码子偏好性。因此，我们在大肠杆菌Rosetta 2(DE3)中产生了TRbeta1，该菌株含有一个过表达对应于七个最罕见大肠杆菌密码子的tRNA的质粒。这导致全长TRbeta1的产量提高，然后我们将其用于电泳迁移率变动分析。我们发现共激活因子TIF2极大地增强了T3占据的TRs与一部分TREs的结合，这表明TRs可能以不依赖RXR的方式从这些TREs激活转录。
