Basic Mechanism of Transcription Elongation by E. coli R

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，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。我们在这个项目中取得的最新成果总结如下： 为了成功地分离和研究延伸和终止中间产物，区分位于延伸或终止途径的RNA聚合酶复合体与那些代表酶与转录本和模板转录后重新结合的产物是非常重要的。 在这项工作中，我们分析了大肠杆菌RNA聚合酶(RNAP)在体外内源转录终止过程中RNA和DNA的释放。为了稳定这个过程的中间产物，我们使用了低离子强度的转录。为了便于它们的分离，我们在追逐它们到终止子之前，通过模板中的生物素基团或酶中的组氨酸标签将其固定在琼脂糖珠上。尽管相当一部分伸长复合体在到达终止子后迅速解体，但仍发现一些RNA和DNA与RNAP有关。我们发现，这些终止的RNA和DNA片段与RNAP形成了二元络合物。二元RNAP/RNA复合体在高盐浓度下不稳定，容易被Greb因子切割，并能够在2-3个核苷酸处对切割产物进行模板无关的延伸。二元复合体的数量依赖于延伸复合体的浓度，这表明它们是RNA/蛋白质重新结合的二次加合物，而不是真正的终止中间体。与RNAP结合的另一部分RNA属于被阻止的复合体，在高盐中长期孵育后不解离。除了RNAP的二元组分和受阻的三元组分被搁置在终止途径之外，我们未能分离到其他不稳定的三元络合物，它们将属于主流途径。

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. The most recent results that we obtained in this project are summarized below: For sucessful isolation and study of elongation and termination intermediates, it's very important to distinguish between RNA polymerase complexes located in the elongation or termination pathways from those that represent the products of the post-transcriptional re-association of the enzyme with transcript and template. In this work we analyzed release of the RNA and the DNA from E. coli RNA polymerase (RNAP) during intrinsic transcription termination in vitro. To stabilize intermediates of the process we used transcription at low ionic strength. To facilitate their isolation, we used immobilization of elongation complexes before chasing them to terminator, on agarose beads through either the biotin group in the template, or the histidine-tag in the enzyme. Although substantial fraction of elongation complexes disintegrated quickly upon reaching the terminator, some of the RNA and DNA were found in association with RNAP. We showed, that these fractions of terminated RNA and DNA formed binary complexes with RNAP. The binary RNAP/RNA complex was labile at high salt concentrations, susceptible to cleavage with factor GreB and capable of template-independent elongation of the cleavage products at 2-3 nucleotides. The amount of the binary complexes was dependent on concentration of elongation complex, which indicated that they were secondary adducts of RNA/protein re-association rather than true termination intermediates. Another fraction of RNA bound to RNAP belonged to the arrested complex, which did not dissociate after prolonged incubation in high salt. Apart from the binary and arrested ternary associates of RNAP lying aside the termination pathway, we failed to isolate previously reported by others unstable ternary complexes, which would belong to the mainstream pathway.