The long-term goal is to understand the mechanism by which peptidyl-prolyl cis/trans isomerases (PPIases) function as molecular switches to regulate gene activity. PPIases catalyze the isomerization of the peptide bond that precedes the cyclic amino acid proline, causing conformational changes that facilitate the folding of newly-synthesized proteins and that regulate the activity of mature proteins by altering their activity or protein- protein interactions. PPIases are found in all organisms, and are best known as the targets of immunosuppressive drugs. However, their normal function in cells is poorly understood. We study a PPIase called Ess1, which is essential for growth in Saccharomyces cerevisiae. Ess1 and its human ortholog, Pin1 (which can substitute for Ess1 in yeast), are implicated in transcription regulation and mitotic cell cycle control. In the pathogenic fungi, Candida albicans and Cryptococcus neoformans, Ess1 is essential for virulence. In humans, misexpression of Pin1 may contribute to cancer and neurodegenerative disease. These findings make clear the importance of prolyl isomerization for cellular function. The goal of the proposed research is to understand the mechanism by which Ess1 recognizes and regulates RNA polymerase II (RNAPII) in eukaryotic cells. Work from this laboratory has shown that Ess1 binds and isomerizes peptidyl-prolyl bonds within the carboxy-terminal domain (CTD) of the large subunit of RNAPII. Ess1 acts by throwing a conformational ?proline switch? thus plays an integral role in specifying the so-called ?CTD code? that helps coordinate the recruitment of protein co-factors to the transcribing RNAPII complex. Ess1-induced conformational changes in the CTD are likely to be important for multiple steps in the transcription cycle. The goal of Aim1 is to understand how Ess1 ?reads the CTD code,? a critical first step in its regulation of the RNAPII transcription cycle. To determine how Ess1 recognizes and binds the CTD a combination of structural, biochemical, biophysical and in vivo studies will be used. The results will be important for understanding how the mammalian ortholog (Pin1) recognizes RNAPII and a variety of other substrates. This information will also be useful for efforts to develop antifungal inhibitors (Ess1) or anti-cancer drugs (Pin1). The goal of Aim2 is to understand how Ess1 ?writes the CTD code? by focusing on its role in transcription elongation. The working hypothesis is that Ess1 controls the recruitment and/or activity of elongation factors, and/or chromatin modifiers, thus regulating RNAPII elongation. This will be tested using biochemical and genomic approaches. These results will be important for two main reasons. First, they will help us understand how non-covalent proline switches (versus covalent modification such as phosphorylation ? an area already heavily studied) regulate recruitment of RNAPII co-factors to the CTD. Second, they will help us understand how Ess1 regulates elongation, a mechanism that is increasingly recognized as critical in gene regulation. To our knowledge, this is the only proposal, and we are the only laboratory, studying the role of this key peptidyl-prolyl isomerase in transcription.
This project proposes to study the structure and function of Ess1, a key enzyme found in organisms ranging from yeast to humans. The proposed studies will use yeast as a model system to determine exactly how Ess1 works. So far, we know that Ess1 plays an important role in the transcription of genes from DNA into RNA, but the detailed mechanisms are not known. Determining these mechanisms is critical to understanding the function of the human enzyme, called Pin1, which has been implicated in cancer and neurodegenerative diseases. This knowledge will be important for understanding what goes wrong with Pin1 in human cells to cause disease. The Ess1 enzyme is also present in pathogenic yeast, which cause systemic fungal infections in patients who have undergone transplants or are immunocompromised (e.g. HIV-infected). The results may help us to identify inhibitors for development into a new class of anti-fungal drugs.
