Proteomics, Macromolecular Complexes, Transcriptional Regulatory Complexes, Cellular Responses to DNA damage
Research in the Ranish lab centers around the development of state of the art proteomics technologies and the application of these technologies to study the function of macromolecular complexes involved in basic biological processes such as transcription and the response to DNA damage. We recently developed a quantitative mass spectrometry approach that can be used to determine the composition of complexes and to detect changes in complex composition (
Figure 1 ). It is based on the use of stable isotope tagging of proteins and mass spectrometry to compare the relative abundances of tryptic peptides derived from suitable pairs of purified or partially purified protein complexes. Application of the technology to study transcription factor complexes from yeast and higher eukaryotes has resulted in the discovery of new transcription factors, and has revealed mechanisms underlying gene regulation during development.
Projects often begin with a discovery driven approach in which macromolecular complexes are isolated and characterized using mass spectrometric techniques. After extensive computational analysis of the data, subsequent studies are performed to validate the results and to test specific hypotheses aimed at gaining a detailed understanding of how the complexes function. In addition to proteomics approaches, we also use genomics, biochemistry, molecular biology and genetics to accomplish these goals.
Research
One major area of research in the lab is to understand how cells control gene expression programs at the level of transcription. Specific gene expression programs underlie basic cellular processes such as the ability to grow, divide, differentiate, and respond to the environment. These programs involve the regulated transcription of thousands of genes by the nuclear RNA polymerases. Presently, our understanding of how cells control and coordinate gene expression programs at the level of transcription is not clear. To elucidate this process, we need to know the composition of gene-specific transcription complexes, which includes identifying the proteins and their post-translational modifications, and how their composition changes in response to cellular and environmental stimuli. Currently, we are addressing this problem by developing methods to isolate gene-specific transcription complexes from cells grown in different conditions, and applying quantitative proteomics approaches to analyze the composition of the complexes.
We are also characterizing novel proteins that were previously identified in a quantitative proteomics screen of RNA polymerase II transcription complexes. One of the recently discovered factors, TFB5, is an evolutionarily conserved component of the general transcription and DNA repair factor TFIIH. Mutations in the gene encoding human TFB5 account for a rare form of DNA repair-deficient tricothiodystrophy disorder. We are investigating the role of TFB5 in transcription and DNA repair processes.
The link between transcription and DNA repair has prompted us to also begin investigating dynamic protein interaction networks that are involved in cellular responses to DNA damage. We are using quantitative proteomics approaches to characterize the composition of complexes that are required for the proper response to DNA damaging agents, and to detect changes in complex composition after exposure to these agents.
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