Mapping polymerase modifications
Scientists at LMU Munich and Helmholtz Zentrum have developed a method for the thorough analysis of protein modifications.
The genetic information stored in our genome is actually silent (i.e. inactive) and must be made to "speak". Like the playback head in a tape recorder, the multisubunit enzyme RNA polymerase II, Pol II for short, runs over the DNA and transcribes the genetic and epigenetic information into RNA. In order to keep the enzyme from working randomly, however, it is dynamically modified at many different points in order to control its activity depending on the situation.
"Phosphorylation makes it possible to influence the activity of the enzyme at 240 different sites," explains Prof. Dirk Eick, the study's last author and head of the Research Unit Molecular Epigenetics at Helmholtz Zentrum München. Together with Prof. Axel Imhof and Dr. Tobias Straub from the Biomedical Center at LMU, and colleagues at LMU’s Gene Center and the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, he and his team have developed a method for simultaneously examining all 240 sites in Pol II. The results have now been published in the science journal “Molecular Cell”.
Mapping of every phosphorylation site
The new work is based on a combination of genetic and mass spectrometric methods. The attachment of a phosphate group to one of its protein subunits alters not only the function of Pol II, but also its mass. “This change in molecular weight can be detected by means of high-resolution mass spectroscopy,” as Axel Imhof explains. However, the large number of potential phosphorylation sites available in the enzyme significantly complicates this task. In order to be able to examine the phosphorylation state of every site in Pol II, artificial recognition sites for protein-fragmenting enzymes were introduced at defined points in each of its subunits. Cleavage of the chains at these pre-determined points gives rise to a set of distinguishable fragments with defined lengths, derived from known locations, which are then subjected to mass spectrometry. Using this approach, the researchers were able to unambiguously assign the mass-spectrometric data to specific sites, and thus to map every single phosphorylation. “By producing genetically modified variants of the regions in question, we can examine each individual phosphorylation,” reveals first author Dr. Roland Schüller from the Helmholtz Zentrum. However, mass spectrometry generates huge amounts of data. “And these data could only be interpreted with the help of complex bioinformatic methods,” says Tobias Straub. The scientists also successfully compared the Pol II modification patterns in humans and in yeast.
“The regulation of the transcription of genes by Pol II is an elementary process in life and deviations in gene regulation are the basis for many human disorders,” as study leader Eick explains. “Research into the phosphorylation pattern at certain times during the transcription cycle is therefore necessary in order to gain an understanding of the underlying mechanisms of gene regulation at the transcriptional level sometime in the future.”
Molecular Cell 2016 (Helmholtz Zentrum/LMU)