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）的两个主要特性。导致稳定延伸复合物形成的途径以及导致基因内和转录末端Pol II解离的机制尚不清楚。区分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体。该方法通过引入核苷酸序列、长度和配对亲和力的变化来评估核酸组分的影响。我们已经证明，8核苷酸RNA:DNA杂交是必要的和充分的形成一个稳定的真核生物EC。此外，我们还观察到RNA:DNA杂交体负向调节Pol II加工的能力，这是以前未知的。这种杂交的双重作用提供了一种机制来控制正确的核酸结构在延伸复合体和Pol II的处理。我们在这个项目中获得的最新成果总结如下：

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.