Niu H, Jacob S T
Department of Pharmacology and Molecular Biology, Chicago Medical School, IL 60064.
Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):9101-5. doi: 10.1073/pnas.91.19.9101.
Previous studies from this laboratory have demonstrated that the enhancer 1 binding factor (E1BF), a Ku-related protein, purified from the serum-enriched cells functions as a positive factor in an RNA polymerase (pol I) transcription system. We have now shown that E1BF purified from the serum-deprived cells (E1BFs) can inhibit rDNA transcription completely in a fractionated extract from the cells grown in serum-enriched medium. The suppression of transcription was overcome by the addition of control E1BF (E1BFc). Immunoprecipitation of purified E1BFs by the anti-Ku monoclonal antibody and addition of the supernatant to the transcription reaction mixture prevented the inhibition significantly, whereas immunoprecipitation with the control mouse IgG did not restore the transcription. The transcriptional repressor activity associated with the final DNA affinity column fractions copurified with E1BF. Neither the amount of E1BF nor its promoter binding activity was altered following serum depletion. E1BFs selectively inhibited the initiation of rDNA transcription. The inhibitory activity of E1BFs was not due to a nonspecific RNase activity. These data suggest that E1BF is post-translationally modified following serum starvation of cells, and that the repressor activity of E1BFs is largely responsible for the down-regulation of pol I transcription in serum-deprived cells.
该实验室之前的研究表明,从富含血清的细胞中纯化得到的增强子1结合因子(E1BF),一种与Ku相关的蛋白,在RNA聚合酶(pol I)转录系统中作为正性因子发挥作用。我们现在已经表明,从血清饥饿细胞中纯化得到的E1BF(E1BFs)能够在富含血清培养基中生长的细胞的分级提取物中完全抑制rDNA转录。通过添加对照E1BF(E1BFc)可克服转录抑制。用抗Ku单克隆抗体对纯化的E1BFs进行免疫沉淀,并将上清液添加到转录反应混合物中,可显著防止抑制作用,而用对照小鼠IgG进行免疫沉淀则不能恢复转录。与最终DNA亲和柱级分相关的转录抑制活性与E1BF共纯化。血清饥饿后,E1BF的量及其启动子结合活性均未改变。E1BFs选择性抑制rDNA转录的起始。E1BFs的抑制活性并非由于非特异性核糖核酸酶活性。这些数据表明,细胞血清饥饿后E1BF发生了翻译后修饰,并且E1BFs的抑制活性在很大程度上导致了血清饥饿细胞中pol I转录的下调。