Ali M, Vedeckis W V
J Biol Chem. 1987 May 15;262(14):6771-7.
The glucocorticoid receptor (GR) from mouse AtT-20 pituitary tumor cells, when transformed using a variety of in vitro protocols, yields a DNA-binding RNA-containing 6 S form. In order to better understand the physiological role of RNA interaction with the transformed GR, we have isolated and purified the putative RNA from AtT-20 cells. [3H]Triamcinolone acetonide-labeled cytosolic GR was transformed, using Sephadex G-25 filtration, to yield the RNA-containing 6 S GR. The transformed 6 S GR was separated on DEAE-cellulose into the 4 S GR (eluting at about 100 mM KCl) while its associated RNA eluted at 0.30-0.45 M KCl. The addition of only these RNA fractions to the 4 S GR can reconstitute 6 S GR as shown on 5-20% sucrose gradients. RNA (0.3-0.45 M KCl fractions) was further purified by hydroxylapatite chromatography, and the bound RNA (eluted at approximately 70 mM PO4(-2)) was then loaded onto preparative 5-20% sucrose gradients to separate RNA on the basis of size (sedimentation rate). A uniform class of RNA sedimenting at 4 S was obtained and then adsorbed to oligo(dT)-cellulose columns. The unbound fraction (poly(A-)) was capable of shifting 4 S GR to 6 S. Using these chromatographic procedures about 90% of the cellular RNA, incapable of reconstituting the 6 S GR from the 4 S form, was eliminated. The 4 S GR was covalently cross-linked with the purified RNA (termed PIVB RNA) using formaldehyde. The resulting cross-linked GR X RNA complexes were shown to sediment at the density of ribonucleoprotein (1.38 g/cm3) in CsCl gradients and at the 6 S position in high salt sucrose gradients. The hydrolysis of PIVB RNA with ribonuclease A prevented the formation of high salt-resistant ribonucleoprotein complexes, indicating that the GR may be in close contact with PIVB RNA. Electrophoresis of the PIVB RNA on 5% agarose-formaldehyde-denaturing gels yielded one major band with a molecular size of approximately 75 bases. It thus appears that an endogenous 4 S RNA (PIVB RNA) of about 25 kDa specifically interacts with the monomeric 4 S GR to yield the 6 S GR.
当使用各种体外实验方案进行转化时，来自小鼠AtT - 20垂体肿瘤细胞的糖皮质激素受体（GR）会产生一种含DNA结合RNA的6S形式。为了更好地理解RNA与转化后的GR相互作用的生理作用，我们从AtT - 20细胞中分离并纯化了假定的RNA。用[³H]曲安奈德丙酮化物标记的胞质GR通过Sephadex G - 25过滤进行转化，以产生含RNA的6S GR。转化后的6S GR在DEAE - 纤维素上分离为4S GR（在约100 mM KCl时洗脱），而其相关RNA在0.30 - 0.45 M KCl时洗脱。如在5 - 20%蔗糖梯度上所示，仅将这些RNA组分添加到4S GR中就能重构6S GR。RNA（0.3 - 0.45 M KCl组分）通过羟基磷灰石色谱进一步纯化，然后将结合的RNA（在约70 mM PO₄⁻²时洗脱）加载到制备型5 - 20%蔗糖梯度上，根据大小（沉降速率）分离RNA。获得了一类沉降系数为4S的均匀RNA，然后将其吸附到寡聚（dT） - 纤维素柱上。未结合部分（poly(A⁻)）能够将4S GR转变为6S。使用这些色谱方法，大约90%的不能从4S形式重构6S GR的细胞RNA被去除。4S GR与纯化的RNA（称为PIVB RNA）使用甲醛进行共价交联。结果表明，所得的交联GR - RNA复合物在CsCl梯度中以核糖核蛋白的密度（1.38 g/cm³）沉降，在高盐蔗糖梯度中在6S位置沉降。用核糖核酸酶A水解PIVB RNA可防止形成高盐抗性核糖核蛋白复合物，这表明GR可能与PIVB RNA紧密接触。PIVB RNA在5%琼脂糖 - 甲醛变性凝胶上进行电泳产生一条主要条带，分子大小约为75个碱基。因此，似乎一种约25 kDa的内源性4S RNA（PIVB RNA）与单体4S GR特异性相互作用以产生6S GR。
