The Architecture of RNA Polymerase III Initiation Complexes

嵌入我们DNA中的遗传信息通过RNA中间体有效地解码成蛋白质。在真核生物中，将DNA忠实地转录成RNA的过程是由三种不同的转录机制进行的，RNA聚合酶I、II和III。每种RNA聚合酶都负责特定基因子集的转录。RNA聚合酶III是专门用于转录参与基本细胞功能的短必需RNA的酶，如trna和5S rRNA。为了有效地转录真核生物基因组，RNA聚合酶I、II和III依赖于不同的转录因子，这些转录因子选择性地识别特定类型的基因，并相应地招募同源RNA聚合酶。在过去的二十年里，真核生物的转录机制已经被广泛地表征：RNA聚合酶II的详细结构图和RNA聚合酶I和III的总体结构已经获得。这些结构信息有助于理解真核生物转录机制的功能和机制。然而，关于类别特异性转录因子招募和协助其同源RNA聚合酶形成转录能力强的起始前复合物的机制，目前的结构信息非常稀缺。因此，转录起始过程仍然不清楚，因此，我们的目标是使用综合结构生物学方法获得Pol III预起始复合物的结构和功能信息。我们关注的是Pol III体系，因为Pol III核心预引发复合物特别稳定。正确组装起始前复合物所需的特定转录因子是Pol III酶稳定相关的亚基，而在Pol II体系中，类似的转录因子是可解离的。为此，我们能够分离和纯化结晶级Pol III核心预起始复合物，使用内源性酵母RNA聚合酶III和重组产生的转录因子。为了研究这些大分子复合物的结构，我们正在整合冷冻电镜和晶体学，这是一种最近使我们能够在结构和功能上表征Pol III核心酶的方法。结构信息将是至关重要的，以了解潜在的机制，控制装配的功能性真核生物预起始复合物，能够准确地启动转录。由于转录起始是一个高度调控的过程，我们的发现将对基因表达调控领域产生深远的影响。此外，由于Pol III起始复合物的组装在癌细胞中通常是一个不受调节的过程，我们的研究结果将为开发和测试基于Pol III转录水平正常化的新抗癌疗法提供机会。此外，Pol III产物已被证明是雷帕霉素靶（TOR）途径控制细胞和生物体生长的重要效应物，因此提出的研究结果可以影响TOR控制的其他重要生物过程，如应激反应和衰老。

The genetic information embedded in our DNA is efficiently decoded into proteins via a RNA intermediate. In eukaryotes, the process of faithfully transcribing DNA into RNA is carried out by three distinct transcription machineries, RNA polymerase I, II and III. Each RNA polymerase is responsible for the transcription of a specific subset of genes. RNA polymerase III is the enzyme devoted to the transcription of short essential RNAs which are involved in fundamental cellular functions, such as the tRNAs and the 5S rRNA. To efficiently transcribe the eukaryotic genome, RNA Polymerase I, II and III rely on distinct sets of transcription factors, which selectively recognise a specific class of genes and accordingly recruit the cognate RNA polymerase. During the last twenty years, the eukaryotic transcription machineries have been extensively characterised: a detailed map of RNA polymerase II structure and the overall architecture of RNA polymerase I and III have been obtained. This structural information has been instrumental in understanding the function and the mechanisms of eukaryotic transcription machineries. Nevertheless, a very scarce amount of structural information is available regarding the mechanisms by which class-specific transcription factors recruit and assist their cognate RNA polymerase to form a transcriptionally competent pre-initiation complex. As a consequence, the process of transcription initiation remains obscure and, for this reason, we are aiming to obtain structural and functional information of Pol III pre-initiation complexes using an integrated structural biology approach. We are focussing on the Pol III system, since Pol III core pre-initiation complexes are particularly stable. Specific transcription factors required for the correct assembly of a pre-initiation complex are stably associated subunits of the Pol III enzyme, whereas in the Pol II system the analogous transcription factors are dissociable. To this end, we were able to isolate and purify crystallization-grade Pol III core pre-initiation complexes, using endogenous yeast RNA Polymerase III and transcription factors produced recombinantly. To study the structures of these large macromolecular complexes, we are integrating cryo-EM and crystallography, an approach that recently enabled us to structurally and functionally characterize the Pol III core enzyme. The structural information will be critical in order to understand the underlying mechanisms which govern the assembly of functional eukaryotic pre-initiation complexes which are able to accurately initiate transcription. As transcription initiation is a highly regulated process, our findings will have a profound impact on the field of gene expression regulation. Additionally, since the assembly of Pol III initiation complexes is a process often deregulated in cancer cells, our findings will provide an opportunity to develop and test new anti-cancer therapies based on normalization of Pol III transcription levels. Furthermore, Pol III products have been shown to act as essential effectors of the target-of-rapamycin (TOR) pathway to control cellular and organismal growth, hence the output of the proposed research can impact other important biological processes controlled by TOR, such as stress response and aging.