Hallick R B, Lipper C, Richards O C, Rutter W J
Biochemistry. 1976 Jul 13;15(14):3039-45. doi: 10.1021/bi00659a016.
A transcriptionally active chromosome has been isolated in highly purified form from choroplasts of Euglena gracilis, It contains chloroplast DNA, DNA-dependent RNA polymerase, and other proteins. Transcription occurs at low levels of endogenous DNA, and is indifferent to high levels of exogenous DNA. RNA chain elongation continues for several hours in vitro, and RNA chain initiation, determined by [gamma-32P]ATP incorporation, is continuous for at least 1 h in vitro. Maximal rates for RNA synthesis require only a divalent cation and the four ribonucleoside triphosphates. Apparent Km values for adenosine triphosphate, cytidine triphosphate, guanosine triphosphate, and uridine triphosphate are 4.0, 0.6, 2.5, and 2.3 muM, respectively. As would be expected for a DNA-dependent RNA polymerase, RNA synthesis is inhibited by actinomycin D. However, rifampicin and streptolydigin, inhibitors of procaryotic RNA synthesis, and alpha-amanitin, an inhibitor of eucaryotic nuclear RNA polymerases II and III, do not inhibt the RNA synthesis reaction. Heparin, which is a potent inhibitor of the initiation of RNA synthesis by a nontemplate bound RNA polymerase, also does not inhibit RNA synthesis. Isolation of transcriptionally active chromosomes should prove to be a useful method to study the mechanism of selective RNA transcription of eucaryotic chromosomes.
已从纤细裸藻的叶绿体中以高度纯化的形式分离出一种转录活性染色体。它含有叶绿体DNA、依赖DNA的RNA聚合酶和其他蛋白质。转录在内源性DNA水平较低时发生，对外源性DNA的高水平不敏感。RNA链延伸在体外持续数小时，通过[γ-32P]ATP掺入确定的RNA链起始在体外至少持续1小时。RNA合成的最大速率仅需要二价阳离子和四种核糖核苷三磷酸。三磷酸腺苷、三磷酸胞苷、三磷酸鸟苷和三磷酸尿苷的表观Km值分别为4.0、0.6、2.5和2.3μM。正如对依赖DNA的RNA聚合酶所预期的那样，RNA合成受到放线菌素D的抑制。然而，原核RNA合成的抑制剂利福平、链霉溶菌素，以及真核细胞核RNA聚合酶II和III的抑制剂α-鹅膏蕈碱，均不抑制RNA合成反应。肝素是一种非模板结合的RNA聚合酶起始RNA合成的有效抑制剂，它也不抑制RNA合成。转录活性染色体的分离应被证明是研究真核染色体选择性RNA转录机制的一种有用方法。
