Eukaryotic RNA polymerase I (Pol I) transcribes ribosomal RNA, a key component of ribosomes. Pol I transcription accounts for the majority of the total RNA in cells, and its upregulation in human cells is a hallmark of cancer while its downregulation is a hallmark of several developmental disorders. Pol I transcription is understudied compared to transcription by Pol II and even Pol III. Our preliminary work suggests fundamental differences between Pol I and Pols II and III that are the basis for this proposal. Our broad long-term objectives are to determine the molecular mechanism of Pol I transcription and how its dysregulation leads to cancer and developmental disorders. There are major gaps in our understanding of (1) the structural organization and architecture of Pol I transcription complexes; (2) the mechanism for how Pol I initiation factors interact with rDNA, which encodes ribosomal RNA; and (3) the molecular function of several key Pol I transcription factors in the activation process. The first rationale for this work is that determining the mechanism and regulation of Pol I transcription will form the molecular basis for understanding how Pol I defects lead to human disease. Our central hypothesis is that Pol I factors use a unique mechanism to carry out transcription and their structure and function is different from the mechanisms governing Pol II and III transcription. The second rationale is that understanding the Pol I transcription mechanism at the most basic and fundamental levels will translate to a better understanding of the connection between Pol I and cancer, leading to new cancer therapeutic strategies. Our proposed research will use a conceptually and technically innovative cross-organismal and interdisciplinary approach that employs a combination of bioinformatic, computational, molecular, biochemical, genetic, genomic, proteomic, and structural methods in the yeast and human cells. Guided by strong preliminary studies, we will test two specific aims: (1) Determine the unique ?coactivator? role of TATA-binding protein (TBP) in Pol I transcription, and (2) Determine the mechanism of Pol I transcription activation. To accomplish these aims, we will use well-established and complementary approaches to identify and map novel Pol I interactions in their native context. We will complement these studies with structural modeling in combination with molecular, genetic, and biochemical functional assays to identify Pol I factor functions conserved from yeast to humans. The proposed research is significant because it will lead to a detailed description of the Pol I transcription mechanism and will provide a conceptual framework for understanding the link between Pol I and human disease. Ultimately, this work will illuminate new avenues for diagnosis, potential interventions, and the development of therapies targeting these novel protein-protein and protein-DNA interactions.
Many human diseases including genetic disorders and cancers are caused by mutations in transcription factors or their dysregulation. The overall goal of this project is to determine the mechanism of transcription by eukaryotic RNA polymerase I (Pol I) and how it is dysregulated in disease. This work will provide fundamental knowledge about the basic molecular mechanism of Pol I transcription and discover key steps that may be targeted in cancer and developmental disorders to enhance health and reduce illness.
