Significant advances have been made in understanding the regulation of eukaryotic gene expression in the past years. The complexity and intricacy inherent in RNA polymerase II-mediated transcription is manifested by the large number of components that facilitate and regulate this process. The advances in this field have been buttressed by convergence of yeast genetics and biochemistry, the development of advanced technologies that expedite protein isolation and identification, the exploitation of structural biology and additional resources gained from the genome project, such as genomics and proteomics. The establishment of well-defined transcription assays has expedited the characterization of the functional role(s) of many of these factors. We are now in a position to analyze the dynamic relationships of these components at the protein:protein and protein:DNA levels. To this end, this proposal contains detailed analyses of several stages of the transcription process including studies of how specific factors modify each stage. This represents an expansion of the ongoing studies in my laboratory regarding the identification and functional role(s) of each of the key components in transcription. As well, this proposal now tackles the regulation of each of the activities of these components. This will define the precise steps and factors that are targeted by moderators that evolved to control the transcription process. Finally, this proposal includes studies of the transcription cycle using natural promoters with natural activators within the context of chromatin. These studies will complement the proposed functional analyses by defining the spacial interrelationships of the transcription factors with respect to each other and with respect to their access to transcription moderators. Our studies on promoter recognition and activation by the general transcription factors of the RNA polymerase II transcription system have broad implications for the mechanism of RNA synthesis in general and will provide the basis for understanding how specific gene transcription factors can modify the transcriptional activity of a particular gene or a set of genes. It is likely that these studies will yield basic principles from which the regulatory mechanisms of class II gene expression can be discerned at the molecular level.