Kermekchiev M, Muramatsu M
Department of Biochemistry, University of Tokyo Faculty of Medicine, Japan.
Nucleic Acids Res. 1993 Feb 11;21(3):447-53. doi: 10.1093/nar/21.3.447.
Extracts obtained from mouse cells growth arrested at stationary phase or under serum starvation exhibit no specific rDNA transcription activity. Experiments with mixed transcriptionally active and inactive whole cell extracts (WCE) obtained from rapidly dividing or growth arrested cells, respectively, demonstrate that rRNA synthesis in vitro can be suppressed by a polymerase I transcription inhibitory activity (PIN), present in inactive extracts. This inhibition effect is not related to increased nuclease activity and affects neither the non-specific Pol I transcription, nor a polymerase II promoter. A comparison of WCE isolated under different growth conditions indicates that PIN changes according to the physiological state of the cell. It reaches a maximal level soon after serum depletion and disappears rapidly when cells are allowed to recover in serum-rich medium. PIN can be clearly demonstrated in WCE but not in nuclear or cytoplasmic extracts and can be also obtained by an additional high salt extraction of nuclei. Furthermore, gel retardation and transcription-in-pellet assays demonstrate that rDNA promoter binding and preinitiation complex stability are similar in active and inactive WCE. This indicates that some later stage(s) of rDNA transcription, rather than the preinitiation complex formation, are attenuated by inactive extracts. Analysis of partially fractionated extracts suggests that PIN is not associated with but can be separated from polymerase I.
从处于静止期或血清饥饿状态下生长停滞的小鼠细胞中获得的提取物没有特异性的核糖体DNA（rDNA）转录活性。分别用从快速分裂或生长停滞的细胞中获得的具有转录活性和无转录活性的全细胞提取物（WCE）进行的实验表明，体外rRNA合成可被无活性提取物中存在的一种聚合酶I转录抑制活性（PIN）所抑制。这种抑制作用与核酸酶活性的增加无关，既不影响非特异性的Pol I转录，也不影响聚合酶II启动子。对在不同生长条件下分离得到的WCE进行比较表明，PIN根据细胞的生理状态而变化。血清耗尽后不久它就达到最高水平，当细胞在富含血清的培养基中恢复时它会迅速消失。PIN在WCE中可以清楚地显示出来，但在核提取物或细胞质提取物中则不然，并且通过对细胞核进行额外的高盐提取也可以获得。此外，凝胶阻滞和沉淀转录分析表明，在有活性和无活性的WCE中，rDNA启动子结合和起始前复合物的稳定性相似。这表明rDNA转录的某些后期阶段，而不是起始前复合物的形成，被无活性提取物所减弱。对部分分级分离的提取物的分析表明，PIN不与聚合酶I相关，但可以与聚合酶I分离。
