Single molecule studies of eukaryotic transcription initiation

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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.