Cell-free genomics reveals fundamental regulatory principles of the Mycobacterium tuberculosis transcription cycle. Froom et al.

CFG_inVitro_transcription_gels.pptx: These data show the radiolabeled transcript output from in vitro transcription reactions performed using purified proteins and linear DNA scaffolds. Figure 4D compares radiolabeled trinucleotide products generated by purified Mycobacterium tuberculosis RNA polymerase in complex with the initiation factor SigA and essential transcription factors (TFs) CarD and RbpA, with and without the essential TF holo-WhiB1 (the holo form of the WhiB1 protein contains a [4Fe-4S]2+ cluster coordinated by four encoded cysteines). Figure 4D demonstrates that adding holo-WhiB1 to the reaction increases the amount of trinucleotide products produced on a linear DNA piece containing the PdesA2 promoter from the M. tuberculosis genome, indicating that holo-WhiB1 activates this promoter. Figures 5D, 5E and 5F compare the influence of the essential M. tuberculosis Nus factors (NusA and NusG) on three different terminators from the M. tuberculosis genome: pitB, icd1, and the 5S rRNA. Comparing the ratiometric intensities of the terminator band versus the "run-off" band (indicative of longer RNA products that were produced by RNA polymerase that did not terminate but processed to the end of the linear DNA fragment) demonstrates that NusG stimulates termination on pitB, while NusA does not; NusA stimulates termination more than NusG on icd1; and NusA and NusG additively stimulate termination on the 5S rRNA terminator.

whiB1_transcription_assay_realtime_fluorescence_measurements.xlsx: These are the raw measurements of real-time fluorescence traces obtained by adding Mtb RNAP holoenzyme, with and without holo-WhiB1, to a pre-incubation mix of all four ribonucleotides (NTPs) and DNA constructs containing variants of PdesA2 with WT or mutant promoter elements driving the expression of the Spinach-mini RNA aptamer that binds to and alters the emission of the fluorescent dye DFHBI. The fluorescence values measured in a control reaction without NTPs were subtracted from each reaction trace, and the value measured at time zero was also subtracted from all values within a given reaction trace, to obtain the background-subtracted and normalized values. These data demonstrate that a minimal GAT motif and intermediate-strength -35 promoter element are each required for WhiB1 to activate transcription of the desA2 promoter, while converting the intermediate-strength -35 to a strong -35 causes WhiB1 to repress transcription.