Abstract: Gene expression requires spatiotemporal coordination of transcription machinery at specific chromatin targets across the vast genomic landscape. How transcription proteins navigate the three-dimensional nuclear space and assemble into molecular machines to shape gene activation remains elusive. In my seminar, I will present fundamental principles governing transcription machinery dynamics. Using live-cell single-molecule imaging in budding yeast, we demonstrate that RNA Polymerase II achieves efficient target search and transcription bursting via spatial confinement, directed by its carboxy-terminal domain, global chromatin organization, and the Mediator complex. Through system-wide analysis of all three RNA polymerase systems, we reveal transient assembly and exchange of transcription complexes, challenging traditional models derived from ensemble and steady-state approaches. Our studies provide an emerging biophysical framework of transcription, offering new mechanistic insights across time and space, with broad implications for cellular function and disease.