The Architecture of RNA Polymerase III Initiation Complexes

RNA 聚合酶 III 起始复合物的结构

• 批准号: BB/K014390/1
• 负责人: Alessandro Vannini
• 金额: $ 64.06万
• 依托单位: Institute of Cancer Research
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK014390%2F1
• 关键词: Architecture; RNA; Polymerase; III; Initiation

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.

嵌入在我们DNA中的遗传信息通过RNA中间体被高效地解码成蛋白质。在真核生物中，将DNA忠实地转录成RNA的过程是由三个不同的转录机制完成的，即RNA聚合酶I、RNA聚合酶II和RNA聚合酶III。每个RNA聚合酶负责特定基因亚集的转录。RNA聚合酶III是一种转录短的必需RNA的酶，参与细胞的基本功能，如tRNAs和5S rRNA。为了有效地转录真核基因组，RNA聚合酶I、II和III依赖于不同的转录因子组，这些转录因子选择性地识别特定类别的基因，并相应地招募同源RNA聚合酶。在过去的二十年里，真核转录机制得到了广泛的研究：获得了RNA聚合酶II的详细结构图和RNA聚合酶I和III的总体结构图。这些结构信息有助于理解真核转录机制的功能和机制。然而，关于类特异性转录因子招募和协助其同源RNA聚合酶形成转录前-起始复合体的机制，结构信息非常稀少。因此，转录起始过程仍然不清楚，因此，我们的目标是使用一种综合的结构生物学方法来获得POLIII预起始复合体的结构和功能信息。我们将重点放在POL III系统上，因为POL III核心预引发络合物特别稳定。正确组装预启动复合体所需的特定转录因子是POL III酶的稳定相关亚单位，而在POL II系统中，类似的转录因子是可分离的。为此，我们使用内源酵母RNA聚合酶III和重组产生的转录因子分离和纯化了结晶级Pol III核心预引发复合体。为了研究这些大分子复合体的结构，我们正在将冷冻EM和结晶学相结合，这一方法最近使我们能够从结构和功能上表征POLIII核心酶。结构信息对于理解控制真核生物功能的预启动复合体组装的潜在机制将是至关重要的，这些真核细胞预启动复合体能够准确地启动转录。由于转录启动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POLIII起始复合体的组装是癌细胞中经常放松调控的过程，我们的发现将为开发和测试基于POLIII转录水平正常化的新的抗癌疗法提供机会。此外，Pol III产物已被证明是雷帕霉素靶标(TOR)途径的基本效应者，以控制细胞和生物的生长，因此拟议的研究成果可以影响TOR控制的其他重要生物过程，如应激反应和衰老。

项目成果

Mechanism of selective recruitment of RNA polymerases II and III to snRNA gene promoters.

• DOI: 10.1101/gad.314245.118
• 发表时间: 2018-05-01
• 期刊: Genes & development
• 影响因子: 10.5
• 作者: Dergai O;Cousin P;Gouge J;Satia K;Praz V;Kuhlman T;Lhôte P;Vannini A;Hernandez N
• 通讯作者: Hernandez N

Molecular mechanisms of Bdp1 in TFIIIB assembly and RNA polymerase III transcription initiation.

• DOI: 10.1038/s41467-017-00126-1
• 发表时间: 2017-07-25
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Gouge J;Guthertz N;Kramm K;Dergai O;Abascal-Palacios G;Satia K;Cousin P;Hernandez N;Grohmann D;Vannini A
• 通讯作者: Vannini A

New tricks for an old dog: Brf2-dependent RNA Polymerase III transcription in oxidative stress and cancer.

• DOI: 10.1080/21541264.2017.1335269
• 发表时间: 2018
• 期刊: Transcription
• 作者: Gouge J;Vannini A
• 通讯作者: Vannini A

RNA polymerase I, bending the rules?

RNA聚合酶I，打破规则？

• DOI: 10.15252/embj.201797924
• 发表时间: 2017
• 期刊: The EMBO journal
• 作者: Jochem L

Structural rearrangements of the RNA polymerase III machinery during tRNA transcription initiation.

• DOI: 10.1016/j.bbagrm.2017.11.005
• 发表时间: 2017-11
• 期刊: Biochimica et biophysica acta. Gene regulatory mechanisms
• 作者: E. Ramsay;A. Vannini