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Structural Studies Of The Transcriptional Coactivator Saga And Of Its Functional Complexes

Reference : PhD Patrick SCHULTZ

Offer publication : April 6, 2016

The transcriptional coactivator SAGA contains 19 different polypeptides for a total molecular weight of more than 1.8 MDa1. This complexity reflects the functional diversity of SAGA in vivo which includes enzymatic functions such as histone acetylation (HAT) or de-ubiquitination, interaction functions with transcriptional activators, general transcription factors such as the general transcription factor TBP as well as nucleosome
recognition functions. The SAGA complex thus forms an interface between transcription activators that recognize specific DNA sequences upstream of gene promoters, chromatin and the transcription pre initiation complex. In order to fully understand SAGA’s mode of action and also its dysfonctioning in some neurodegenerative diseases, structural data is required on precisely characterized functional intermediates.

The only available 3-D model of human and yeast SAGA was obtained in the host laboratory by electron microscopy2-4. Several subunits have been localized by immunolabelling or
through the analysis of deletion mutants which allowed us to propose a first model for the quaternary architecture of SAGA. Important progresses were made in SAGA purification and we are in a unique position to make significant progresses in struicture determination by cryo electron microscopy.

The research project will be divided into four parts :

1- High resolution structure determination of yeast SAGA Advanced cryo electron microscopy (CryoEM) methods will be used to visualize frozen hydrated SAGA molecules. The objective is to reach a resolution close to 5 Å by numerical analysis of a large number of molecular images. Exceptional cryo electron microscopes are available at the host laboratory as well as all expertise and computing resources needed for data analysis.

2- Interaction of TATA binding Protein TBP The general transcription factor TBP interacts with SAGA and regulates pre-initiation complex assembly. We can reconstitute this interaction and our objective is to determine the interaction site of TBP with SAGA to understand its links with transcriptional
activators.

3- Action of transcriptional activators. A large number of transcriptional activators interacts with SAGA and recruits the coactivator complex on the promoter of the gene to be transcribed. The interaction mode of these factors with the Tra1 subunit of SAGA and the mode of activation signal
transduction are still largely unknown. Structural information will allow us to locate the interaction site of activators and identify conformational changes within SAGA.

4- Interaction with chromatin. The binding mode of SAGA to the core nucleosome and to chromatin fragments will be studied in order to better understand the structural basis of SAGA’s function in the chromatin decondensation associated to transcription initiation. Purified SAGA will be incubated with preparations of mono-, di- and tetra-nucleosomes and cryoEM will be used to visualize these complexes.

5- Transcription initiation. Functional complexes implicated in SAGA-dependant transcription initiation will be reconstituted on specific promoters containing activator binding sites. The objective of this part will be to analyze an activation transcription complex and to determine its molecular structure.

- WISHED SKILLS : The candidate needs to have a strong background in molecular and structural biology. He (She) should master computing tools and some prior knowledge in image analysis would useful. The candidate needs to integrate structural and functional information from different origins
and therefore needs to have an ability to synthesize.

EXPERTISES WHICH WILL BE ACQUIRED DURING THE TRAINING : The scientific project is centered on the structural analysis of transcriptional co-activators and will provide the candidate with a deep knowledge on the regulatory mechanisms that guide gene expression. The candidate will be exposed to structural biology methods and in particular protein purification, cryo electron microscopy and image analysis. He (She) will combine cryo electron microscopy data and atomic models from crystallography and will investigate conformational changes and macromolecular dynamics.

Your application

Application Deadline : Dec. 31, 2016

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