A1 Analyses of the RNA polymerase I (Pol I) machinery: specific transcriptional mechanisms, comparison with other RNA polymerases and structural determination of Pol I complexes | H. Tschochner, J. Griesenbeck, P. Milkereit
RNA polymerase I (Pol I) is a specialized enzyme which transcribes ribosomal RNA (rRNA) genes in all eukaryotes investigated so far. Pol I transcription is distinguished by specific DNA cis-elements and trans-acting factors as well as the usage of a specific chromatin template. The aim of this project is to unravel specific functional and structural features of the Pol I machinery on a molecular level and to get more insights into the mechanism of Pol I dependent RNA synthesis. Well-defined in vitro systems, which were established in the first funding period will now be used to investigate how certain subunits, domains and/or posttranslational modifications of the Pol I machinery are involved in specific steps of the transcription cycle. 

A4 Coordination of rRNA gene transcription initiation and termination in chromatin | G. Längst
Project A4 focuses on the establishment and functional role of promoter-terminator DNA loops in rDNA regulation. In WP1 we will study the interplay between NoRC, pRNA and TTF-I in promoter recognition and nucleosome positioning. In WP2 we will functionally characterize the chromatin structure at the rRNA gene terminator and test whether this structure is a prerequisite for efficient transcription termination and rDNA looping. In WP3 we will study the dynamics of rDNA chromatin structure and activity during the cell cycle, monitoring the dynamics of chromatin structure and factor binding. We will establish genomically tagged TTF-I, UBF, NoRC and Chd proteins to reveal genomic locations, isolate complexes and to reveal the cell cycle dependent dynamics of these proteins

A5 Compositional analysis of ribosomal RNA gene chromatin in distinct transcriptional states | J. Griesenbeck, P. Milkereit, H. Tschochner
The essential process of ribosomal RNA (rRNA) gene transcription in the nucleolus of eukaryotic cells occurs in the context of chromatin. Our project aims to define rRNA gene chromatin, in its composition, structure and function. In the last funding period we have further developed a technique for the native isolation of rRNA gene chromatin from S. cerevisiae, providing a detailed description of chromatin at different functional ribosomal DNA (rDNA) elements. Results obtained with the biochemically purified rDNA chromatin were verified by methods analyzing rDNA chromatin composition in vivo. In the next funding period we will use the combination of these approaches to obtain a detailed molecular description of dynamic rDNA chromatin transitions in different physiological situations. We expect to deepen our insights in the structure-function relationship between chromatin and transcription.

A6 Transcript elongation and RNA processing/export in Arabidopsis | K. Grasser
The aim of this project is to elucidate possible interactions between ongoing transcription and mRNA processing and export in the Arabidopsis model. Mutants deficient in different transcript elongation factors (TEFs) will be examined for mRNA splicing defects. Affected genes are analysed in more detail to identify whether changes in chromatin structure correlate with splicing alterations.  In the second part, the interplay of transcript elongation and plant mRNA export will be studied to elucidate a possible link with the transcript elongation complex. Since in plants mRNA export proteins (i.e. export adaptors) are diversified, it will be analysed to which extent they are functionally redundant or have adopted specific roles in the export of mRNAs from the nucleus.

A7 Dynamics of transcriptional complexes and interplay of the archaeal RNA polymerase with RNA processing proteins | D. Grohmann
We will employ single-molecule fluorescence spectroscopy in conjunction with site-specific fluorophore engineering schemes and biochemical approaches to unravel the structure-function-dynamic relationship within archaeal and also eukaryotic RNAPI and RNAPIII transcription systems, which only recently became available in a recombinant form suitable for single-molecule interrogation. Here, we will especially focus on mechanisms of transcription initiation. Furthermore, we would like to characterise the influence of the archaeal RNA chaperone Hfq on RNA synthesis as these processes most likely occur simultaneously in vivo.

A2 (finished): Structure and function of RNA polymerase I and initiation factor Rrn3 | P. Cramer
The first step in eukaryotic ribosome biogenesis is the production of the ribosomal RNA precursor by RNA polymerase I (Pol I). Pol I is a 14-subunit multi-protein complex with a molecular weight of 600 kDa. The regulation of Pol I and ribosome biogenesis controls cell growth. Despite its importance, the structure of Pol I and the mechanism of class I promoter-specific transcription initiation remain unknown. The long-term goal of this project is to provide the structural basis of Pol I transcription, and Pol I-specific transcription initiation, to understand key switches that control cell growth. In the past, we have determined crystal structures of Pol I subcomplexes A14/43 and A43/34.5 (Geiger et al., Mol. Cell 2010) and the hybrid structure of Pol I that used cryo-electron microscopy, modelling, and biochemical probing, to unravel the functional architecture of the complex (Kuhn, Cell 2007). During the first funding period of this SFB, we propose to determine the X-ray crystal structure of the complete yeast Pol I (Aim 1), and to determine the structure and function of the Pol I-specific initiation factor Rrn3, including its interaction with Pol I (Aim 2). In collaboration with other teams in the SFB, we will also improve crystals of the initiation complex of the archaeal Pyrococcus furiosus polymerase, provide assistance to teams who aim to crystallize factors involved in ribosome biogenesis, and provide help with structure-based prediction and interpretation of site-directed mutagenesis.

A3 (finished): Structure function relationships in archaeal transcription machinery | M. Thomm
The archaeal transcription machinery is the evolutionary precursor and an excellent simplified model of the more complex eukaryotic transcription systems. The reconstituted RNA polymerase (RNAP) from Pyrococcus furiosus was a useful tool for the elucidation of major functions of key loops in the active centre of RNAP and the archaeal system was also helpful to unravel a function of the linker region of TFB and of the clamp coiled coil domain of RNAP in open complex formation. Eukaryotic RNAP subunits were shown to functionally replace subunits in the archaeal RNAP, and the archaeal subunit P in WT sequence and a mutated form of subunit H were able to complement deletion mutants of the general eukaryotic RNAP subunits Rpb12 and of the C-terminal domain of subunit Rpb5 in yeast. In this project we aim to analyze the architecture and dynamics of the preinitiation complex (PIC) in more detail in particular with respect to the location and function of different functional elements of TFB that are inserted in the RNA polymerase cleft in a TFIIB-RNA polymerase II cocrystal. We are specifically interested in the transitions from i) closed to open complexes and from ii) open to early elongating complexes. Furthermore, we continue to determine the location of TFE on the RNA-polymerase during initiation and elongation, both in vivo and in vitro. Another target of investigation is transcriptional proofreading of the archaeal RNA polymerase. Reconstitution of the archaeal RNA polymerases allows us to study deletion and point mutants in the trigger loop (TL) to define its contribution to internal cleavage activity of the RNA polymerase, transcriptional fidelity as well as a possible role of TL in interacting with elongation factor TFS, which is located in cocrystals of Pol II and TFIIS close to the TL. These studies will contribute to a deeper understanding of the mechanism of transcription and of the evolution of the three eukaryotic RNA polymerases from a single archaea-like precursor.