Lights, machines, action! Watching gene expression unfold at the single-molecule level.

Macromolecular machines, such as RNA polymerase and the ribosome, are part of complex interconnected processes. Due to their complexity and dynamic nature, coupled processes are difficult to capture, making single-molecule imaging best suited to watch the involved machines in action. Recent advances push single-molecule techniques toward more complex in vitro systems, employing multiple colors and even watching machines in live cells. In this review, we highlight recent studies that employ single-molecule fluorescence microscopy to investigate complex molecular processes relevant to gene expression using complex in vitro reconstitutions or novel approaches to study gene expression in living cells.