Rice G A, Chamberlin M J, Kane C M
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
Nucleic Acids Res. 1993 Jan 11;21(1):113-8. doi: 10.1093/nar/21.1.113.
Elongation complexes of RNA polymerase II, RNA-DNA-enzyme ternary complexes, are intermediates in the synthesis of all eukaryotic mRNAs and are potential regulatory targets for factors controlling RNA chain elongation and termination. Analysis of such complexes can provide information concerning the structure of the catalytic core of the RNA polymerase and its interactions with the DNA template and RNA transcript. Knowledge of the structure of such complexes is essential in understanding the catalytic and regulatory properties of RNA polymerase. We have prepared and isolated complexes of purified RNA polymerase II halted at defined positions along a DNA template, and we have used deoxyribonuclease I (DNAse I) to map the interactions of the polymerase with the DNA template. DNAse I footprints of three specific ternary complexes reveal that the enzyme-template interactions of individual elongation complexes are not identical. The size of the protected region is distinct for each complex and varies from 48 to 55 bp between different complexes. Additionally, the positioning of the protected region relative to the active site varies in different complexes. Our results suggest that RNA polymerase II is a dynamic molecule and undergoes continual conformational transitions during elongation. These transitions are likely to be important in the processes of transcript elongation and termination and their regulation.
RNA聚合酶II的延伸复合物，即RNA-DNA-酶三元复合物，是所有真核生物mRNA合成过程中的中间体，并且是控制RNA链延伸和终止的因子的潜在调控靶点。对此类复合物的分析能够提供有关RNA聚合酶催化核心结构及其与DNA模板和RNA转录本相互作用的信息。了解此类复合物的结构对于理解RNA聚合酶的催化和调控特性至关重要。我们制备并分离了沿DNA模板在特定位置停滞的纯化RNA聚合酶II复合物，并且使用脱氧核糖核酸酶I（DNAse I）来绘制聚合酶与DNA模板的相互作用图谱。三种特定三元复合物的DNAse I足迹显示，各个延伸复合物的酶-模板相互作用并不相同。每种复合物的保护区域大小各异，不同复合物之间在48至55碱基对之间变化。此外，保护区域相对于活性位点的定位在不同复合物中也有所不同。我们的结果表明，RNA聚合酶II是一个动态分子，在延伸过程中经历持续的构象转变。这些转变可能在转录延伸和终止过程及其调控中发挥重要作用。
