Project Summary The goal of this project is to contribute to understanding of the biochemical and biophysical mechanisms by which gene expression is regulated. In particular, we seek to understand the operation of the molecular machinery that regulates production of mRNA by DNA transcription and the relationship between the composition and structures of the macromolecular complexes that make up that machinery and their functions. In this project, we have developed and applied a powerful approach to quantitatively defining the dynamic molecular mechanisms of transcription and transcription regulation in vitro. Instead of studying populations of molecules, we use multi-wavelength single-molecule fluorescence methods to directly visualize in vitro the RNA polymerase and associating regulatory proteins simultaneously on hundreds of isolated individual DNA molecules in multiplex. We propose continuing studies that apply this approach to elucidating dynamic molecular regulation mechanisms of the messenger RNA synthesis machinery from an example bacterium (E. coli) and from a model eukaryote (the budding yeast S. cerevisiae). In both systems, we have chosen for study transcription factors and regulatory activities that are general mechanisms which act during transcription of many or most protein-coding genes in the organism. Our studies will advance the field by focusing not on single regulatory factors in isolation but on understanding the emergent properties of systems in which multiple regulatory factors act together. Our immediate goals are to use multi-wavelength single-molecule fluorescence methods in vitro to 1) test the hypothesis that bacterial RNA polymerase elongation complexes are specialized by alternative modes of σ70 binding and determine how specialization dictates different combinatorial regulation by elongation factors NusA and NusG; 2) reveal the mechanisms that determine the outcome of the bacterial post- termination complex; and 3) define the pathways by which selected general elongation factors compete and cooperate for recruitment to eukaryotic RNA polymerase II elongation complexes.