Basic Mechanism of Transcription Elongation by E. coli R

大肠杆菌 R 转录延伸的基本机制

• 批准号: 6763559
• 负责人: MIKHAIL KASHLEV
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6763559
• 关键词: DNA directed RNA polymerase; Saccharomyces cerevisiae; enzyme activity; fungal genetics; fungal proteins; genetic transcription; microorganism culture; oligonucleotides

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.

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。延伸复合物的稳定性和活性中的延伸因子的作用和Pol II本身的作用之间的区别需要一个简单的，“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。我们在该项目中获得的最新结果总结如下： 为了成功地分离和研究延伸和终止中间体，区分位于延伸或终止途径中的RNA聚合酶复合物与代表酶与转录物和模板的转录后再结合产物的RNA聚合酶复合物是非常重要的。 在这项工作中，我们分析了RNA和DNA从E. coli RNA聚合酶（RNAP）在体外内源性转录终止过程中的作用。为了稳定该过程的中间体，我们在低离子强度下使用转录。为了促进它们的分离，我们在将它们追踪到终止子之前，通过模板中的生物素基团或酶中的组氨酸标签将延伸复合物固定在琼脂糖珠上。虽然相当大一部分的延伸复合物在到达终止子后迅速解体，但发现一些RNA和DNA与RNAP相关。我们发现，这些部分的终止RNA和DNA与RNAP形成二元复合物。二元RNAP/RNA复合物在高盐浓度下是不稳定的，易受因子GreB切割的影响，并且能够在2-3个核苷酸处不依赖于模板地延伸切割产物。二元复合物的量依赖于延伸复合物的浓度，这表明它们是RNA/蛋白质再缔合的次级加合物，而不是真正的终止中间体。另一部分RNA结合RNAP属于被捕的复合物，在高盐条件下长时间孵育后不解离。除了位于终止途径旁边的RNAP的二元和被捕的三元缔合物之外，我们未能分离出先前由其他人报道的不稳定的三元复合物，其将属于主流途径。