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

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 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 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. Overall design: RNA samples were prepared by incubating a mixture of purified M. tuberculosis (Mtb) RNA polymerase core enzyme, co-purified Mtb sigma factor A (SigA) and Mtb RbpA, purified Mtb CarD, sonicated Mtb genomic DNA, ribonucleotides, RNase inhibitor, and pyrophosphatase for 1 hour - 5 hours at 37ºC, in the presence or absence of transcription factors of interest (Mtb CRP, Mtb WhiB1, Mtb NusG, Mtb NusA). The aim was to identify TSSs or TTSs where these TFs can individually affect transcription. Similarly-derived in vitro RNA from the E. coli genome was spiked into all reactions at equivalent amounts to control for different amounts of total RNA across samples. 5' or 3' ends of the RNA were then enriched and sequenced. Core RNAP only samples were used to filter out non-SigA-dependent (i.e. non-specific) TSSs from the RNAP-SigA holoenzyme TSS samples. Genomic DNA input samples were used to filter out any TTSs that may be enriched due to non-random fragmentation of the genomic DNA input from the RNAP-SigA holoenzyme TTS samples.