The core transcription machinery is the ultimate target of many signal transduction and developmental pathways and regulation of transcription is one of the key steps in control of cell growth, differentiation and development. Because of the complexity of the RNA polymerase II (Pol II) core machinery, its inherent flexibility and dynamic behavior, there are large gaps in understanding the mechanisms of regulated assembly of the machinery, DNA unwinding, start site scanning, and initiation. The long term goals of this project are to determine the mechanisms of RNA Pol II transcription initiation and how these mechanisms are utilized as targets for gene regulation. The rationale for this work is that determining the mechanisms used in initiation and its regulation will form the molecular basis for understanding defects in transcription disorders leading to many types of human disease. The objectives of this application are to determine the overall architecture of the complete Pol II preinitiation complex (PIC) and open complex (OC) states, with a focus on understanding mechanisms used by general transcription factors that act in DNA melting and start site scanning. These studies will utilize biochemical, molecular genetic, structural, and biophysical methods to examine the pathway of transcription initiation by S. cerevisiae Pol II, an experimental system that can utilize a powerful mix of molecular genetics and biochemistry. Since the transcription machinery is well-conserved, gene regulatory mechanisms in yeast are nearly always used in mammalian cells. To understand the mechanism of OC formation, biochemical approaches pioneered in the last grant period to determine the structural arrangement of large complexes will be used to map the architecture of the complete PIC and OC. New approaches and recent technical advances will be used to examine the architecture of the general factor TFIIH and the function of its many subdomains, taking full advantage of yeast molecular genetics to examine in vivo function. The mechanisms of DNA unwinding and transcription start site scanning will be visualized using both bulk biochemical and innovative single molecule approaches. These new approaches will reveal rate-limiting steps in the initiation pathway as well as new functions for the general factors. Our proposed research is significant because it will lead to a vertical advance in understanding the pathway of transcription initiation by Pol II, new roles for the general transcription factors in this process and lay the foundation for understanding the mechanism of transcriptional regulators that act directly on the core transcription machinery.