Cell-free genomics reveals fundamental regulatory principles of the Mycobacterium tuberculosis transcription cycle

• Cell-free genomics (CFG) maps direct RNAP and transcription factor effects genome wide • CFG reveals intrinsic promoter and terminator rules in M. tuberculosis • Removing cellular noise reveals direct promoter targets of TF CRP and essential TF WhiB1 • Essential TFs NusA and NusG control distinct intrinsic termination mechanisms Cell-free genomics (CFG) maps direct RNAP and transcription factor effects genome wide CFG reveals intrinsic promoter and terminator rules in M. tuberculosis Removing cellular noise reveals direct promoter targets of TF CRP and essential TF WhiB1 Essential TFs NusA and NusG control distinct intrinsic termination mechanisms Tiers of gene regulation govern cellular life. The intrinsic activities of RNA polymerase (RNAP) constitute a primary tier, while direct modulation by accessory transcription factors (TFs) constitutes a secondary tier. Cellular signaling cascades and feedback loops generate tertiary and higher-order tiers. Dissecting gene regulation requires distinguishing direct TF targets at the genome scale from indirect network effects. A major obstacle is the lack of tools to interrogate transcription machineries from difficult-to-culture microbes—such as pathogens, commensals, and environmental species—at the genome scale. Here, we introduce cell-free genomics (CFG), an empirical approach that identifies the direct targets of RNAP and TFs and systematizes their transcriptional effects. We demonstrate the efficacy of CFG by characterizing global and essential transcription initiation (CRP and holo-WhiB1) and elongation-termination factors (NusA and NusG) from the deadly pathogen, Mycobacterium tuberculosis . CFG expands our understanding of transcription principles and is broadly extensible to other perturbations and diverse species.