Transcription swells chromosomesin vitro

Chromosomes provide the basic genetic instructions for all living cells, and their reconstitution with steady-state gene expression remains to be established. The in vitro reconstitution from a single chromosome would reveal basic insights into its native conformation, accessibility, and functionality, with key implications for the design of synthetic cells (1). Here, we report on transplanting the native chromosomes from Escherichia coli cells into steady-state microfluidic compartments for cell-free gene expression (2). Upon cell-free expression, we observe a marked swelling of the 4.6 Mbps-long DNA molecules, responding abruptly to transcription inhibition by a ~2.7-fold global compaction. Extracting chromosomes with native proteins decorating the DNA, we find the compaction counterbalanced. High-resolution optical mapping of the genome organization with chromosome-bound proteins, including the RNA polymerases (RNAP) and ribosomes, and their cell-free dynamics on DNA proposes the bacterial structural maintenance of chromosomes complex MukBEF (3-6) as a source for local DNA compaction, guiding the transcription-induced swelling and global compaction by macromolecular crowding. Taken together, molecular machines may form an intricate balance between genome-scale swelling and point-like DNA compaction, suggesting proteinated chromosomes as active gels.