This project utilized 3D orbital tracking and a newly developed fluorescent labeling strategy that allows simultaneous visualization of pre-mRNA and transcription factors inside of living yeast cells. This allowed us to follow the fate of individual eukaryotic pre-mRNA molecules as they undergo transcription in real time and enable complete kinetic characterization of the initiation, elongation and release of individual RNA molecules as well as single molecule temporal correlation of transcription factor binding to DNA. In previous work, due to the rapid photobleaching of cells, 10-20 measurements were averaged together to determine transcriptional kinetics. With 3D orbital tracking, the information garnered in three previous experiments on two separate microscopes will be available in a single cell measurement at a 100x times faster sampling rate.