Single molecule studies of eukaryotic transcription initiation

控制基因表达水平对任何真核细胞都是至关重要的，了解这种控制的分子机制是本研究计划的主要目的。虽然转录延伸的结构研究使我们对转录延伸的基本步骤有了很好的机制理解，但转录起始的结构生物学已被证明更加困难，因为涉及许多因素和转录起始复合物固有的灵活性。出于这个原因，结合交联实验、电子显微镜、质谱、晶体学研究或小角度x射线散射等信息的混合结构方法将对促进我们的理解变得重要。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提供实时动力学信息，另一方面可以用于获取结构信息。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（NPS）来获得真核生物转录起始的结构和机制。首先，我们将克服以往真核生物转录NPS实验的局限性，开发利用酶标记策略制备酵母RNA聚合酶II （Pol II）复合物，将染料分子附着在各种选定的位点上。有了这项技术，我们将揭示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的各种截断突变体来确定各个结构域的机制作用。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中的序列特异性。此外，我们将在体外组装功能性转录启动复合物，包括高纯度的酵母Pol II， TBP， TFIIB， TFIIE， TFIIF和TFIIH以及启动子DNA。通过这些配合物，我们将研究从开放到封闭配合物的转变。在这里，单分子FRET研究将再次有助于确定关键构象变化，以达到机理理解。特别是，我们将设计smFRET实验来确定TFIIH介导的DNA熔化机制。构象变化将实时研究使用smFRET与一个或两个FRET对同时进行。对于从CC到OC的过渡机制重要的构象将使用NPS进一步研究。因此，拟议的研究将为真核生物转录的起始提供机制见解。

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.