TRANSCRIPTION ELONGATION BY RNA POLYMERASE II

高结构稳定性和催化活性是RNA聚合酶II（Pol II）的进行性延伸复合物的两个主要性质。导致形成稳定的延伸复合物的途径和引起Pol II在基因内和转录终止子处解离的机制还不清楚。延伸因子在延伸复合物稳定性和活性中的作用与Pol II本身的作用之间的差异需要一个简单的、最小的体外系统。在这个项目中，我们开发了一种新的技术，绕过了蛋白质因子启动转录和获得延伸复合物的需要。该技术涉及使用纯化的核心Pol II和合成的RNA和DNA寡核苷酸直接组装延伸途径中的中间体。该方法允许通过寡核苷酸的序列、长度和配对亲和力对寡核苷酸引入变化来评估核酸组分的影响。我们已经证明，8个核苷酸的RNA：DNA杂交是必要的，足以形成一个稳定的真核EC。此外，我们已经观察到以前未知的能力的RNA：DNA杂交负调节Pol II持续合成能力。杂交体的这种双重作用提供了一种控制延伸复合物中正确核酸结构和Pol II持续合成能力的机制。

High structural stability and catalytic activity are the two principal properties of the processive elongation complex of RNA polymerase II (Pol II). The pathway leading to the formation of a stable elongation complex and the mechanisms causing dissociation of Pol II within the genes and at transcription terminators are not well understood. Differentiation between the role of elongation factors in elongation complex stability and activity and that of the Pol II itself required a simple, minimal in vitro system. In this project, we develop a novel technique that bypasses the need for protein factors to initiate transcription and obtain the elongation complex. This technique involves the direct assembly of intermediates in the elongation pathway using purified core Pol II and synthetic RNA and DNA oligonucleotides. This method allows to assess the impact of nucleic acids components by introducing changes to the oligonucleotides through their sequence, length, and pairing affinity. We have shown that the 8 nucleotides RNA:DNA hybrid is necessary and sufficient for the formation of a stable eukaryotic EC. In addition, we have observed the previously unknown ability of the RNA:DNA hybrid to negatively regulate Pol II processivity. This dual role of the hybrid provides a mechanism for the control of a correct nucleic acid architecture in the elongation complex, and Pol II processivity.