Project (Christian Dienemann - Elisa Oberbeckmann)
Structural biology and in vitro systems

We are investigating the molecular mechanisms and systemic principles of genome transcription and regulation using a combination of structural biology with functional genomics and bioinformatics in human and yeast cells. We offer several exciting projects in these areas and will recruit highly motivated and talented PhD students with experimental or computational backgrounds. As part of the IMPRS-GS, our laboratory can offer PhD projects in (A) bottom-up in vitro reconstitutions using purified proteins and Next Generation Sequencing (Project leader: Elisa Oberbeckmann) as well as (B) structural biology of gene transcription and chromatin using cryo-electron microscopy (cryo-EM) (Project leader: Christian Dienemann). Please find below additional information regarding our recent and ongoing work relating to the PhD projects.

A : Chromatin architecture can regulate transcriptional activity. Therefore, we aim to understand which proteins influence chromatin architecture to which extent. The most important players are ATP-dependent chromatin remodelers, certain transcription factors and cohesin. In vivo, many of these factors are either redundant or essential. Thus, it is inherently difficult to disentangle their individual contributions and direct activity in vivo. To solve this problem, we set up a bottom-up approach in which each protein can be tested individually or in combination (1,2). We reconstitute chromatin from a plasmid library containing the entire yeast genome and purified histone octamers. After incubation of chromatin with the protein of interest, the protein activity is measured genome-wide with MNase-seq and MicroC (in collaboration with the Oudelaar lab). The project involves genome engineering by CRISPR/Cas9, endogenous and recombinant protein purification from human or insect cells, Next-Generation Sequencing techniques, bioinformatics and potentially cryo-electron tomography. Thus, a strong background in protein biochemistry or NGS is of advantage.

B: Transcription by RNA polymerase II (Pol II) is regulated at many steps throughout the transcription cycle of a gene. We aim to understand the molecular mechanisms behind transcription regulation by reconstituting transcription complexes at different stages of the cycle in vitro, and determine their three-dimensional structures by cryo-EM. The processes of pre-initiation complex assembly, promoter-proximal Pol II pausing and transcription through a nucleosome are of particular interest as these steps are major regulatory events during gene transcription (3). For that, we purify all components of the human transcription machinery and reconstitute transcription in the test tube, either in a promoter-dependent manner or by extension of an existing RNA primer. These samples can then be used for structural and biochemical analyses. A typical project involves protein expression in bacteria and insect cells, protein purification by various chromatography methods as well as gradient-based complex preparation methods. Further, we use gel-based assays for monitoring RNA production by Pol II-containing transcription complexes and eventually cryo-electron microscopy and single-particle image analysis to determine atomic structures of transcription complexes. Therefore, a strong background in protein biochemistry and/or structural biology (X-ray crystallography or cryo-EM) is of advantage.

In our research group we also offer PhD projects in computational and systems biology:
https://www.uni-goettingen.de/de/application/558700.html





Homepage Research Group
http://www.mpinat.mpg.de/cramer



Publications:

1) Quillian et al., In vitro reconstitution of chromatin domains. Biorxiv 2023
2) Oberbeckmann et al., Ruler elements in chromatin remodelers set nucleosome array spacing and phasing. Nature Communications 2021
3) Osman and Cramer. Structural Biology of RNA Polymerase II Transcription: 20 Years On. Annu Rev Cell Dev Biol. 2020