MOLECULAR MECHANISM OF TRANSCRIPTIONAL ACTIVATION

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The long-term focus of this project is to understand the enzymatic mechanisms that underlie the response of RNA polymerase II to transcriptional activator proteins. These proteins regulate RNA synthesis in all cells and play a fundamental role in the control of cell growth and differentiation. Our recent efforts have concentrated on a template-associated protein kinase, DNA-PK, that phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II at about the same time as RNA synthesis begins. We have purified the kinase to homogeneity and have identified its active components. Remarkably, the activity of the purified kinase is strongly stimulated by certain DNA-bound transcriptional activator proteins. This property suggests that the kinase may provide a means of transducing signals from transcriptional activators to the catalytic portion of the transcription apparatus. We propose experiments to better understand the mechanisms by which kinase activity is regulated, and to more clearly delineate the role of the kinase in transcription. Specific aims include: 1. Defining the functional role of kinase subunits. The kinase has a catalytic subunit and two regulatory subunits. We will study how the regulatory subunits affect kinase activity, and determine how the three subunits contact each other, the DNA, and the peptide substrate of the reaction. 2. Studying the interaction of the kinase with transcriptional activator proteins. We will determine how heat shock transcription factor and a fragment of yeast GAL4 stimulate kinase activity and will search for additional factors that may work by the kinase mechanism. 3. Determining the role of template-associated kinase in basal and activated transcription. We will test whether the presence of the kinase affects initiation, promoter clearance, or processivity. We will also test whether RNAP II phosphorylation functions as an energy-dependent proofreading step in initiation. 4. Developing novel probes of RNA polymerase II large subunit C-terminal domain (CTD) structure and function. We will map the contacts that the CTD makes with other transcription factors, generate novel anti-CTD reagents for use in vivo and in vitro, and identify functions of the CTD other than those related to phosphorylation.