The broad, long-term objectives of this proposal are to determine at a molecular level, the mechanism of transcription initiation by RNA Polymerases II and III. These studies will lead to a better understanding of the transcription machinery and how it is regulated. This is important because the transcription machinery is the ultimate target of many signal transduction, cell cycle, and developmental pathways. Understanding the fundamental process of gene control will provide a basis for understanding many types of diseases such as cancer and syndromes involving DNA repair defects which can result from aberrant gene regulation, or from mutations in the transcription machinery. The specific aims of this project are designed to study through molecular, biochemical and genetic methods, the mechanisms of transcription initiation and reinitiation utilized by RNA Polymerases II and III. The role of novel factors, which interact with the TFIIA terminal domain in preinitiation complexes (PICs), will be identified. The mechanism of Pol II transcription reinitiation will be investigated with a focus on the role of the mediator factor and identification of a form of Pol II used in the reinitiation reaction. The role of several distinct mediator complexes in gene regulation will be studied. The role of the core domain of the Pol III factor BRF will be elucidated, and compared to the role of the homologous region of the Pol II factor TFIIB. Finally, the target of the zinc ribbon domains in TFIIB and Brf will be identified, comparing the role of these domains in Pol II and III transcription.