The long-term objective of my laboratory is to understand the molecular mechanisms by which Pol I transcription is regulated in response to physiological and pathological stimuli. In pursuit of this goal we have developed biochemical assays aimed at dissecting the molecular events that lead to the activation of RNA polymerase I transcription upon viral infection. In the past funding period we have discovered a novel kinase activity that plays a seminal role in the stimulation of RNA polymerase I transcription by SV40 large T antigen. Specifically, we have demonstrated that stimulation of Pol I transcription requires the phosphorylation of UBF, a key pol I-specific transcription factor, by a large T antigen associated kinase. This data is consistent with the hypothesis that interaction of a novel large T antigen-kinase complex with Pol I transcription factors is a critical step necessary for both the stimulation of RNA polymerase I transcription and cellular transformation. In accordance with this hypothesis, in specific aim 1, I propose to map the kinase-binding domain and determine the transcriptional properties of large T antigen mutants that have lost the ability to bind to the kinase. These mutants will be further tested in transformation assays such as focus formation, anchorage-independent growth and growth in low serum to determine whether kinase binding and stimulation of rDNA transcription are required for cellular transformation.
In aim 2, I propose to identify the sites in UBF that are phosphorylated by the protein kinase and determine their functional role in activation of ribosomal DNA transcription.
In aim 3, I describe a biochemical approach for the purification and identification of the kinase activity bound to large T antigen that is responsible for the phosphorylation of UBF.
In aim 4, I propose to test the physiological role of the kinase in pol I transcription and determine whether large T antigen-induced alterations in kinase activity are required for cellular transformation. These studies will provide important insights into the mechanistic basis of cell transformation by viral oncogenes.
| Zhai, W; Comai, L (2000) Repression of RNA polymerase I transcription by the tumor suppressor p53. Mol Cell Biol 20:5930-8 |
| Zhai, W; Comai, L (1999) A kinase activity associated with simian virus 40 large T antigen phosphorylates upstream binding factor (UBF) and promotes formation of a stable initiation complex between UBF and SL1. Mol Cell Biol 19:2791-802 |
