All eukaryotic organisms share the complex need for intricate control of gene expression according to the needs of the cell. This is achieved largely in part by dynamic, precise positioning of nucleosomes at gene promoters, which share a defined chromatin architecture characterized by a nucleosome depleted region (NDR) surrounded by modified nucleosomes. The boundary of the NDR is defined by an upstream -1 nucleosome and a downstream +1 nucleosome. The gene promoter contains necessary sequence elements that have been well established to control transcription initiation such as transcription factor binding sites, the TATA box, and the necessary transcription start sites. It has been reported that the nucleosome can obstruct these sequence motifs serving as a barrier to transcription machinery and its positioning provides an important role in regulating gene expression. Nucleosomes are positioned by chromatin remodeling enzymes that use energy from ATP hydrolysis for nucleosome translocation. Many gene promoters recruit multiple remodelers from the sub-classes (SWI/SNF, INO80, ISWI, and CHD) and transcription factors that direct the necessary transcription machinery. However, the kinetics of how these multiple factors that can all slide nucleosomes work at the same gene promoter remains elusive with existing methods. This proposal is based on my preliminary data designing a real-time reporter of +1 nucleosome position to elucidate how multiple remodelers coordinate transcription initiation using the model organism Saccharomyces cerevisiae. Here, I propose a time- resolved system to observe the kinetically separable steps of reconstituted +1 nucleosome movement on native sequences in the context of sequence specific and general transcription factor binding. In Aim 1, I will use DNA from the promoter of the inducible gene HIS3 to reconstitute a +1 nucleosome FRET construct that will report +1 nucleosome positioning over time. In Aim 2, I will add purified chromatin remodeling enzymes to my nucleosome construct and measure the temporal window of promoter accessibility caused by multiple, opposing remodelers, testing the hypothesis that the push and pull by different remodelers are responsible for promoter accessibility. Additionally, I will image the same construct in the presence of whole cell extracts that contain all the native cell components for comparison with purified remodelers. In Aim 3, to measure the functional impact of promoter accessibility, I will test the hypothesis that nucleosome dynamics control the binding of sequence specific transcription factor GCN4 and the general transcription factor TFIID. These experiments provide a direct reporter of +1 nucleosome positioning kinetics at single molecule resolution. Overall, this work will provide unprecedented insight into the multifaceted kinetic interplay of major chromatin and transcription components at a eukaryotic gene promoter for the transition from repressed to active chromatin.