Mirkovitch J, Darnell J E
Rockefeller University, New York, New York 10021.
Mol Biol Cell. 1992 Oct;3(10):1085-94. doi: 10.1091/mbc.3.10.1085.
The assembly of an RNA polymerase II initiation complex at a promoter is associated with the melting of the DNA template to allow the polymerase to read the DNA sequence and synthesize the corresponding RNA. Using the specific single-stranded modifying reagent KMnO4 and a new genomic sequencing technique, we have explored the melted regions of specific genes in genomic DNA of whole cells or of isolated nuclei. We have demonstrated for the first time in vivo the melting in the promoter proximal transcribed region that is associated with the presence of RNA polymerase II complexes. An interferon-inducible gene, ISG-54, exhibited KMnO4 sensitivity over approximately 300 nucleotides downstream of the RNA initiation site in interferon-treated cells when the gene was actively transcribed but not in untreated cells where the gene was not transcribed. The extent of KMnO4 modification was proportional to transcription levels. The KMnO4 sensitivity was retained when nuclei were isolated from induced cells but was lost if the engaged polymerases were further allowed to elongate the nascent RNA chains ("run-on"). The sensitivity to KMnO4 in isolated nuclei was retained if the run-on incubation was performed in the presence of alpha-amanitin, which blocks progress of engaged polymerases. A similar analysis identified an open sequence of only approximately 30 bases just downstream of the start site of the transthyretin (TTR) gene in nuclei isolated from mouse liver, a tissue where TTR is actively transcribed. This abrupt boundary of KMnO4 sensitivity, which was removed completely by allowing engaged polymerases to elongate RNA chains, suggests that most polymerases transcribing this gene paused at about position +20. The possibility of mapping at the nucleotide level the position of actively transcribing RNA polymerases in whole cells or isolated nuclei opens new prospects in the study of transcription initiation and elongation.
RNA聚合酶II起始复合物在启动子处的组装与DNA模板的解链相关，以使聚合酶能够读取DNA序列并合成相应的RNA。利用特异性单链修饰试剂高锰酸钾（KMnO4）和一种新的基因组测序技术，我们探索了全细胞或分离细胞核的基因组DNA中特定基因的解链区域。我们首次在体内证明了与RNA聚合酶II复合物存在相关的启动子近端转录区域的解链。干扰素诱导基因ISG - 54在干扰素处理的细胞中，当该基因被积极转录时，在RNA起始位点下游约300个核苷酸范围内表现出对KMnO4敏感，而在未处理的未转录该基因的细胞中则不敏感。KMnO4修饰的程度与转录水平成正比。从诱导细胞中分离出细胞核时，对KMnO4的敏感性得以保留，但如果让结合的聚合酶进一步延长新生RNA链（“连续转录”），则敏感性丧失。如果在α - 鹅膏蕈碱存在下进行连续转录孵育，分离细胞核对KMnO4的敏感性会保留，α - 鹅膏蕈碱会阻断结合的聚合酶的进程。类似的分析在从小鼠肝脏分离的细胞核中，在转甲状腺素蛋白（TTR）基因起始位点下游仅约30个碱基处鉴定出一个开放序列，小鼠肝脏是TTR活跃转录的组织。这种KMnO4敏感性的突然边界通过让结合的聚合酶延长RNA链而被完全消除，这表明转录该基因的大多数聚合酶在大约 +20位置处暂停。在全细胞或分离细胞核中在核苷酸水平上绘制活跃转录的RNA聚合酶位置的可能性为转录起始和延伸研究开辟了新前景。
