Hodo H G, Sahasrabuddhe C G, Plishker M F, Saunders G F
Nucleic Acids Res. 1980 Sep 11;8(17):3851-64. doi: 10.1093/nar/8.17.3851.
Nucleosomes prepared from human placental nuclei and Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyl transferase EC.2.7.7.6) form stable initiation complexes. This property is utilized as a probe of nucleosome structure. RNA polymerase initiation has been studied on purified nucleosomes, nucleosome cores, and nucleosomal DNA. The affinity of E. coli RNA polymerase for both nucleosome cores and monomers was 5-6 fold less than found for nucleosomal DNA. No difference in apparent initiation Km was found between cores and mononucleosomes. This suggests that initiation does not preferentially occur on the DNA tails of nucleosomes. Once initiated and allowed to form nascent RNA, these complexes are very stable to ionic strength changes. Under conditions in which free enzyme is inactivated with rifampicin, the enzyme in the complex retains activity as demonstrated by its ability to transcribe and reinitiate on both nucleosomes and free DNA. These complexes can be well resolved from free nucleosomes on preparative polyacrylamide gels and both can be eluted from gels for analysis of proteins and DNA sequence complexity. Studies using (125I) labelled nucleosomes show that histones are retained in the initiation complex, and are not dissociated by the enzyme during initiation.
从人胎盘细胞核和大肠杆菌DNA依赖性RNA聚合酶（核苷三磷酸：RNA核苷酸转移酶EC.2.7.7.6）制备的核小体形成稳定的起始复合物。这一特性被用作研究核小体结构的探针。已经对纯化的核小体、核小体核心颗粒和核小体DNA上的RNA聚合酶起始进行了研究。大肠杆菌RNA聚合酶对核小体核心颗粒和单体的亲和力比对核小体DNA的亲和力低5至6倍。在核小体核心颗粒和单核小体之间未发现明显的起始Km差异。这表明起始并非优先发生在核小体的DNA尾部。一旦起始并允许形成新生RNA，这些复合物对离子强度变化非常稳定。在利福平使游离酶失活的条件下，复合物中的酶保留活性，这可通过其在核小体和游离DNA上转录和重新起始的能力得到证明。这些复合物在制备性聚丙烯酰胺凝胶上可与游离核小体很好地分离，并且两者都可从凝胶上洗脱下来，用于分析蛋白质和DNA序列复杂性。使用（125I）标记核小体的研究表明，组蛋白保留在起始复合物中，并且在起始过程中不会被酶解离。
