Project Summary A central concern of the present post-genomic era of biology is understanding the chemical and physical mechanisms by which gene expression is regulated. Appropriate activation and repression of particular genes is necessary for maintaining normal cell function and is required for executing the programs of cell differentiation that are essential to the development of multicellular organisms. Collectively, gene regulatory systems are the “molecular brain” of the cell that allow it to respond appropriately to environmental stimuli. Many cancers and other diseases result from deranged gene regulation. In this research 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 directly visualize the RNA polymerase and associated regulatory proteins on isolated single DNA molecules, following the progression of the molecular machinery through its different states in real time while simultaneously observing the transcription reaction itself. Such direct visualization is made possible by a multi-wavelength single-molecule fluorescence microscopy approach we call CoSMoS (colocalization single-molecule spectroscopy). In this application, we propose applying the CoSMoS approach to elucidating the dynamic mechanisms of selected processes involved in regulation of transcription initiation and elongation using both bacterial and eukaryotic RNA polymerases and regulatory proteins in transcription reactions in vitro. Our goals are: 1) Reveal how two different secondary channel binding proteins exert their regulatory functions through a single shared target site on RNA polymerase. 2) Elucidate the mechanisms by which bacterial elongation complexes are loaded with and regulated by general elongation factors NusA and NusG. 3) Reveal recruitment and competition within sets of general elongation factors in yeast RNA polymerase II elongation complexes.