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Single molecule studies of eukaryotic transcription initiation

真核转录起始的单分子研究

基本信息

批准号：
286540087
负责人：
Professor Dr. Jens Michaelis
金额：
--
依托单位：
Institut für Biophysik
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2016
资助国家：
德国
起止时间：
2015-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/286540087?language=en
关键词：
Single molecule studies eukaryotic transcription

项目摘要

Controlling the level of gene expression is of utmost importance for any eukaryotic cell and understanding the molecular mechanisms underlying this control is the main aim of this research proposal. While structural studies of transcription elongation have given us a very good mechanistic understanding of essential steps during transcription elongation, structural biology of transcription initiation has proven to be more difficult, due to the many factors involved and the inherent flexibility of transcription initiation complexes. For this reason, hybrid structural approaches combining information from e.g. cross-linking experiments, electron microscopy, mass-spectrometry, crystallographic studies or small angle x-ray scattering will become important to advance our understanding. In this respect single molecule fluorescence studies provide an excellent tool, since they can on one hand give information on real time dynamics and on the other hand be used for obtaining structural information. In the proposed experiments we will use single molecule fluorescence and the previously developed Nano Positioning System (NPS) to gain structural and mechanistic insight into eukyaryotic transcription initiation.We will, first, overcome limitations in previous NPS experiments of eukaryotic transcription by developing strategies to prepare yeast RNA polymerase II (Pol II) complexes with dye molecules attached to various selected sites using enzymatic labeling strategies. With this technique at hand we will then unravel the mechanistic role of TFIIF and TFIIE in controlling the position of the downstream DNA during transcription initiation. To this end we will use various truncation mutants of TFIIF to identify the mechanistic roles of the individual domains. We will also investigate different promoter sequences to determine how TFIIF controls sequence specificity in transcription start site selection.Moreover, we will assemble functional transcription initation complexes in-vitro, consisting of highly purified yeast Pol II, TBP, TFIIB, TFIIE, TFIIF and TFIIH together with promoter DNA. With these complexes we will investigate the transition from the open to the closed complex. Here, single molecule FRET studies will again be instrumental to determine key conformational changes in order to reach a mechanistic understanding. In particular, we will design smFRET experiments to determine the mechanism of the DNA melting mediated by TFIIH. Conformational changes will be investigated in real-time using smFRET with one or two FRET pairs simultaneously. Conformations important for the mechansim of the transition from the CC to the OC will be further investigated using NPS.The proposed research will therefore give mechanistic insight into the initiation of eukaryotic transcription.

控制基因表达水平对于任何真核细胞都至关重要，了解这种控制的分子机制是本研究提案的主要目的。虽然转录延伸的结构研究已经给了我们一个非常好的机制理解转录延伸过程中的基本步骤，转录起始的结构生物学已被证明是更加困难的，由于涉及的许多因素和转录起始复合物的固有灵活性。出于这个原因，混合结构方法结合信息，例如交联实验，电子显微镜，质谱，晶体学研究或小角度X射线散射将变得重要，以促进我们的理解。在这方面，单分子荧光研究提供了一个很好的工具，因为它们一方面可以给出真实的时间动力学的信息，另一方面可以用于获得结构信息。在拟议的实验中，我们将使用单分子荧光和先前开发的纳米定位系统（Nano Positioning System，Nano Positioning System），以获得结构和机制的洞察真核转录initiation.We将，首先，克服在真核转录的Nano Positioning实验的局限性，通过开发策略，以制备酵母RNA聚合酶II（Pol II）与染料分子连接到各种选定的网站，使用酶标记策略的复合物。有了这项技术在手，我们将揭开TFIIF和TFIIE在转录起始过程中控制下游DNA位置的机制作用。为此，我们将使用TFIIF的各种截短突变体来鉴定各个结构域的机械作用。我们还将研究不同的启动子序列，以确定TFIIF如何控制转录起始位点选择的序列特异性。此外，我们将在体外组装功能性转录起始复合物，由高度纯化的酵母Pol II，TBP，TFIIB，TFIIE，TFIIF和TFIIH与启动子DNA一起组成。有了这些复合物，我们将研究从开放复合物到封闭复合物的过渡。在这里，单分子FRET研究将再次有助于确定关键的构象变化，以达到一个机械的理解。特别是，我们将设计smFRET实验，以确定TFIIH介导的DNA熔解的机制。将使用同时具有一个或两个FRET对的smFRET实时研究构象变化。构象的重要性，从CC到OC的过渡mechanism将进一步研究使用EST-PCR。因此，拟议的研究将提供真核生物转录的启动机制的见解。