Subsequent projects

Prof. Dr. Joachim Griesenbeck
University of Regensburg
Fakultät für Biologie -
Institut für Biochemie, Genetik und Mikrobiologie


Prof. Dr. Hinrich Boeger
University of California, Santa Cruz
Molecular Cell & Developmental Biology

Structural analysis of single gene molecules by electron and atomic force microscopy

In previous collaborations, we have investigated the mechanism of transcriptional activation of individualgenes in the eukaryotic model organism S. cerevisiae (hereafter called yeast) (Boeger et al., 2008; Brown et al., 2013; Hamperl et al., 2014a, 2014b). To this end we have developed an experimental method for the isolation of molecules of defined genes from yeast chromosomes. This has allowed us to investigate both the protein composition of individual genes in different functional states by mass spectrometry, and to analyse the nucleosome configurations of single gene molecules by electron microscopy. Both approaches have greatly advanced knowledge and understanding of the structural and compositional dynamics of defined chromatin domains; and novel insights into the mechanistic relationship between chromatin structure and transcription were thus obtained.

We wish to continue our collaboration with the goal to further advance electron microscopic (EM) techniques in both laboratories, and to explore the possibility of atomic force microscopy (AFM) for the structural analysis of single gene molecules. AFM microscopy will be performed in collaboration with Holger Schmidt (Electrical Engineering, UCSC); and we would like to include Dr. John van Noort (University of Leiden), an expert on AFM and magnetic force microscopy, who is interested in the acquisition of our chromatin ring isolation method for his research on the elastic properties of chromatin fibers. The hope is that AFM will provide an easier and more efficient experimental tool for the structural analysis of single gene molecules than EM.

References:

Boeger, H., Griesenbeck, J., and Kornberg, R.D. (2008). Nucleosome retention and the stochastic nature of promoter chromatin remodeling for transcription. Cell 133, 716–726.
Brown, C.R., Mao, C., Falkovskaia, E., Jurica, M.S., and Boeger, H. (2013). Linking stochastic fluctuations in chromatin structure and gene expression. PLoS Biol. 11, e1001621.
Hamperl, S., Brown, C.R., Garea, A.V., Perez-Fernandez, J., Bruckmann, A., Huber, K., Wittner, M., Babl, V., Stoeckl, U., Deutzmann, R., et al. (2014a). Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae. Nucleic Acids Res. 42, e2.
Hamperl, S., Brown, C.R., Perez-Fernandez, J., Huber, K., Wittner, M., Babl, V., Stöckl, U., Boeger, H., Tschochner, H., Milkereit, P., et al. (2014b). Purification of specific chromatin domains from single-copy gene loci in Saccharomyces cerevisiae. Methods Mol. Biol. Clifton NJ 1094, 329–341.

 

Primary project: Molecular mechanism of gene activation: Chromatin transition at the yeast PHO5 promoter


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