Original in vitro transcription gels for 'Cell-free genomics reveals fundamental regulatory principles of the Mycobacterium tuberculosis transcription cycle'

These data show the radiolabeled transcript output from in vitro transcription reactions performed using purified proteins and linear DNA scaffolds. Experiment 1 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). Experiment 1 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. Experiment 2 compares 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.