Thomas P, Davies P, Griffiths K
Biochem J. 1977 Aug 15;166(2):189-98. doi: 10.1042/bj1660189.
The characteristics of initiation of RNA synthesis and the elongation of RNA chains on rat ventral-prostate chromatin by RNA polymerase B were investigated by two methods. 1. Initiation was carried out under low-salt conditions with three ribonucleoside triphosphates, and elongation was begun in the absence of reinitiation by the addition of the fourth ribonucleoside triphosphate and increasing the salt concentration. 2. Stable initiation complexes were formed by preincubation of enzyme with template at 37 degrees C, elongation was started by the addition of all four ribonucleoside triphosphates and reinitiation or spurious RNA synthesis was prevented by rifamycin AF/013. The latter method gave more reliable results. The dependence of those parameters on the androgenic status of the animal was studied. During the first 24h after castration, elongation was mainly affected, whereas after 72h a smaller number of initiation sites for RNA polymerase B on chromatin was evident. Considerable diurnal variations in the various parameters were observed. Changes in the relative concentrations of the chromatin-associated proteins were also observed after castration. In the rat ventral-prostate gland androgenic steroids may not only influence one stage of the transcriptional process, but may affect many factors involved in the control of gene expression.
采用两种方法研究了RNA聚合酶B在大鼠腹侧前列腺染色质上起始RNA合成及延伸RNA链的特性。1. 在低盐条件下,用三种核糖核苷三磷酸进行起始反应,然后通过添加第四种核糖核苷三磷酸并提高盐浓度,在无重新起始的情况下开始延伸反应。2. 通过在37℃下将酶与模板预孵育形成稳定的起始复合物,添加所有四种核糖核苷三磷酸开始延伸反应,并用利福霉素AF/013防止重新起始或假RNA合成。后一种方法得到的结果更可靠。研究了这些参数对动物雄激素状态的依赖性。去势后的头24小时内,延伸反应主要受到影响,而72小时后,染色质上RNA聚合酶B的起始位点数量明显减少。观察到各种参数存在显著的昼夜变化。去势后还观察到染色质相关蛋白相对浓度的变化。在大鼠腹侧前列腺中,雄激素类固醇不仅可能影响转录过程的一个阶段,还可能影响许多参与基因表达调控的因素。