Gene transcription is the first step in the expression of the genetic information and a focal point for cellular regulation. Our goal is to understand the molecular mechanisms of gene transcription and the principles of genomic regulation in eukaryotic cells. We use integrated structural biology and complementary functional studies to unravel the three-dimensional and functional architecture of large macromolecular complexes involved in transcription. We also develop functional genomics methods and computational approaches to unravel the cellular mechanisms of genomic regulation. These efforts led to a first molecular movie of transcription and provided insights into gene-regulatory cellular networks. Together, these efforts shape the emerging fields of genome biology and molecular systems biology. Our aim is to understand the functional genome as a regulatory network based on the underlying structural and molecular mechanisms.
Gene Transcription and RNA Polymerases
In eukaryotes, gene transcription is carried out by three related RNA polymerases. RNA polymerase (Pol) I and Pol III produce ribosomal and transfer RNAs, respectively, whereas Pol II transcribes protein-coding genes and produces mRNA, which serves as the template for protein synthesis. RNA polymerases are the endpoint of signal transduction pathways, and their regulation underlies cell growth and differentiation. RNA polymerases are very large enzymes, consisting of 12-17 protein subunits with a total molecular weight of 0.5-0.7 Megadalton (Vannini and Cramer, Mol. Cell 2012). The polymerases assemble with many factors into large transient transcription machineries of changing composition. Polymerase-associated factors enable the polymerases to recognize different promoters and to transcribe different classes of genes, to receive different regulatory signals, to direct the co-transcriptional processing of RNA transcripts, and to couple transcription to changes in chromatin. Therefore, RNA polymerases are not only the key enzymes of gene expression but also the central coordinators of nuclear events.
Integrated Structural Biology of Gene Transcription
To elucidate the mechanisms of transcription in vitro, we determine three-dimensional structures of RNA polymerases in complex with nucleic acid substrates and protein factors. X-ray crystallography allows atomic structure determination of very large and asymmetric macromolecular complexes. In addition, we use protein crosslinking coupled to mass spectrometry and single-particle cryo-electron microscopy, and often combine different structural biology methods with the use of molecular modelling. Such an integrated structural biology approach elucidates the architecture of large and transient assemblies. We have used this approach to obtain many polymerase complex structures, which we assembled into a first movie of Pol II transcription (Cheung and Cramer, Cell 2012). A key intermediate of Pol II transcription initiation (Sainsbury et al., Nature 2012) and a major part of the gene-regulatory coactivator complex Mediator was also structurally resolved recently (Lariviere, Plaschka et al., Nature 2012). We have recently also obtained the crystal structure of Pol I, providing a starting point for analysis of a second eukaryotic transcription system (Engel et al. Nature 2013). In the future, we will extend these studies to resolve regulated polymerase complexes and to understand how the structure and function of the transcription machineries evolved.
Molecular Systems Biology and Genomic Regulation
To systematically investigate how the genome is expressed and regulated in vivo, we study transcription and RNA regulation in the context of living cells. Recent advances in functional genomics now allow us to (a) monitor all transcription activity in cells by next-generation sequencing of newly synthesized RNA (4tU-seq), to (b) map the bindings sites of regulatory protein factors over the genome (ChIP-seq), and to (c) map the location of regulatory RNA-binding factors over the transcriptome (PAR-CLIP). By combining these techniques and evaluating the obtained systemic data with a computational biology approach, often in close collaboration with expert groups such as the groups of Johannes Soeding, Julien Gagneur, and Achim Tresch, we uncover principles of genome transcription and its regulation. Recent achievements in this area include the mapping of transcription factors over the yeast genome (Mayer, Lidschreiber, Siebert et al., NSMB 2010), the development of "dynamic transcriptome analysis" to measure both mRNA synthesis and degradation rates genome-wide (Miller, Schwalb, Maier et al., Mol. Syst. Biol. 2011), an analysis of global mRNA degradation to investigate how cells buffer the levels of their mRNA transcripts (Sun, Schwalb et al. Mol. Cell 2013), and the global analysis of transcriptome surveillance by selective termination of non-coding RNA synthesis (Schulz, Schwalb et al. Cell 2013).