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To develop and respond to the environment, every living cell needs to retrieve and translate the genetic information stored in DNA. This fundamental process is called gene expression and needs to be tightly regulated and coordinated for obvious reasons. The first step, where DNA is copied into RNA, is called transcription and is carried out by a universally conserved protein enzyme called RNA polymerase (RNAP). The general theme of research in our team is the regulation of transcription by protein and non-coding RNA factors. Transcription is a fundamental, yet incompletely understood process. Because of its central role, the bacterial transcription machinery is also a prime drug target. High resolution structures of RNAP core as well as RNAP elongation complexes have revolutionized our understanding of transcription and allowed us to interpret transcription at the molecular level. This groundwork paved the way to address more complex questions regarding the regulation of transcription. Transcriptional regulation impacts every aspect of biology including a growing list of human diseases. Transcriptional pausing, a temporary interruption of transcription, plays a major role in regulating gene expression in pro- and eukaryotes. A full and mechanistic understanding is crucial and requires a combination of structural and biochemical studies. This is where our team is contributing.
Currently we focus on three major aims:
1. How do protein transcription factors modulate the rate of transcription?
2. How do non-coding RNA molecules modulate transcription rates?
3. What is the role of RNA polymerase dynamics and conformational changes during transcription?

Through financial support from start-up grants including the Marie Curie Career Integration Grant, the team started work on all three aims over the past four years. This allowed us to produce preliminary data, which in turn resulted in successful research grant applications, that give us now the freedom to focus completely on the questions outlined above.
The initial period of the project was used to setup purification and biochemical assays to characterize functional complexes of RNA polymerase in complex with protein and RNA transcription factors. At the same time, we transitioned from using X-ray crystallography as our primary method to obtain high-resolution structural information to single particle cryo-EM (electron cryo-microscopy). Given the recent advances in cryo-EM this was an important decision, because obtaining high resolution structures has now become more straight-forward for us.

Aim 1 – Role of protein transcription factors:
A long-standing problem in the bacterial transcription field has been the role of conserved protein transcription factors like NusA, NusG or the Gre factors. NusA is known to stimulate transcriptional pausing and termination, whereas NusG is known to prevent pauses, stimulate termination and might be involved in the coupling of transcription and protein synthesis. The Gre factors are involved in transcriptional fidelity and in the rescue of stalled or arrested RNAP molecules.
Work on protein transcription factors has been our main focus and we have made progress on several projects in this category. All complexes we are currently working on can be purified to homogeneity, are biochemically active and have been used for data collection at different high-end electron microscopes at the host institute (IGBMC Illkirch, France) and other research centers in Europe (EMBL Heidelberg, Germany; ESRF Grenoble, France; Biozentrum Basel, Switzerland; NeCEN Leiden, Netherlands). We have been successful in our transition from X-ray crystallography to single particle cryo-EM, have obtained the necessary know-how, and have setup the computational resources to process and interpret EM data in the team.
We have recently published our first major finding: a high-resolution EM reconstruction of a functional RNAP elongation complex bound by NusA allowed us to shed light on the role of this protein. NusA has been studied for over 40 years and plays a variety of roles including its stimulatory effect on transcriptional pausing. The reconstruction explains how catalysis is inhibited and provided an explanation for the role of NusA. The results have been published in the Journal Molecular Cell (Guo et al., Mol Cell 2018) and were also featured on the cover of the journal.
Similarly, we have encouraging reconstructions of other complexes and are in the process of interpreting them.

Aim 2 – role of RNA as a transcription factor:
RNA is not just a passive carrier of information but has also been implemented in playing an active, regulatory role. We are focussing on several non-coding RNA transcripts that influence transcription in cis. Here we also collaborate with Prof. Renée Schroeder from the University of Vienna, who has identified a large number of potential RNA regulators.
To do this, the team had to setup methods for in vitro transcription to be able and produce large quantities of RNA for biochemical and structural studies. RNA can now be produced and functional complexes can be purified. We have recently obtained our first EM reconstruction that confirmed the presence of all the ligands including the regulatory RNA.

Aim 3 – role of RNAP dynamics and conformational changes in transcription:
Here the initial goal was to setup an experimental system to study RNAP dynamics and conformation by single molecule FRET. We pursued two different approaches in parallel: labelling of RNAP with fluorophores using an enzymatic approach and labelling using non-natural amino acids. Although we made a lot of progress, our focus has shifted because EM reconstructions of functional complexes provide a wealth of information on the conformational freedom of various transcription complexes.

Taken together, the Marie Curie Career Integration Grant was a critical component to get the team started, produce preliminary data and allowed us to apply for competitive funding opportunities on the national (Agence Nationale de la Recherche) and international level (Starting Grant of the European Research Council).

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Life Sciences
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