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)的两个主要性质。导致稳定伸长复合体形成的途径以及导致POL II在基因内和转录终止子上解离的机制尚不清楚。区分伸长因子在伸长复合体稳定性和活性中的作用和POL II本身的作用需要一个简单、最小的体外系统。在这个项目中，我们开发了一种新的技术，它绕过了蛋白质因子启动转录和获得伸长复合体的需要。这项技术包括使用纯化的核心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产物。这种方法允许通过对寡核苷酸的序列、长度和配对亲和力的改变来评估核酸成分的影响。我们已经证明，8个核苷酸的RNA：DNA杂交是形成稳定的真核EC的必要条件和充分条件。此外，我们还观察到了先前未知的RNA：DNA杂交对POL II加工能力的负调控能力。杂交体的这种双重作用为控制延伸复合体中正确的核酸结构和POL II的加工性提供了一种机制。

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.