Original in vitro transcription gels for 'Cell-free genomics reveals fundamental regulatory principles of the Mycobacterium tuberculosis transcription cycle'
Description
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.
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Steps to reproduce
In vitro abortive transcription initiation assays were performed at 37 °C as previously described. PdesA2 promoter was prepared using PCR amplification from Mtb genomic DNA. Assays with the PdesA2 promoter were performed in WhiB1 transcription buffer (10 mM Tris-HCl, pH 8.0, 100 mM K-Glutamate, 10 mM MgCl2, 50 μg/mL BSA, 1 mM DTT and 2mM TCEP, pH 8.0). 50 nM of Mtb RNAP core was incubated with 250 nM of σA–RbpA or σA–RbpA–WhiB1 for 10 minutes at 37 °C to form holoenzyme. CarD (2 μM) and PdesA2 DNA template (10 nM) were then added to both holoenzyme mixtures and incubated at 37 °C for another 10 minutes to form open promoter complexes. Abortive initiation assays were initiated on PdesA2 with the ApU dinucleotide primer (250 μM), [α-32P]GTP (1.25 μCi) and unlabeled GTP (50 μM). After 20 minutes, 2X Stop buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol) was added to stop the reaction. Samples were analyzed on a 23% denaturing PAGE (19:1 acrylamide: bis acrylamide, 7M urea, 1X TBE, pH 8.3) for 1.25 hours at 800 V, and the gel was exposed on a Storage Phosphor Screen and imaged using a Typhoon PhosphoImager (GE Healthcare). Intensity of the trinucleotide products was quantified using ImageJ. Intrinsic terminators were cloned downstream of a cytidine-less halt cassette in plasmid pAC70 and in vitro transcription templates were amplified. HoloRNAP–σA–RbpA (200 nM) was incubated with template DNA (10 nM) and CarD (500 nM) for 15 min at 37 °C in transcription termination buffer (10 mM Tris-HCl pH 8.0, 100 mM KGlu, 10 mM MgOAc, 1 mM DTT, 5 µg/mL BSA). To initiate transcription, the complex was incubated with ATP + GTP (both at 16 µM), UTP (2 µM), and 0.1 µL/reaction [α-32P]UTP for 15 min at 37 °C to form a halted complex at U26. Transcription was restarted by adding a master mix containing NTP mix (A + C + G + U), heparin, and either Nus factor buffer (for the “no TF” condition), NusA, NusG or NusA + NusG at a final concentration of 150 µM (each NTP), 10 µg/mL (heparin), and 1 µM each Nus protein at 23 °C. The reaction was allowed to proceed for 30 min, followed by a “chase” reaction in which all 4 nucleotides were added to a final concentration of 500 µM each. After 10 minutes, 2X Stop buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol) was added to stop the reaction. Samples were analyzed on an 8% denaturing PAGE (19:1 acrylamide: bis acrylamide, 7M urea, 1X TBE, pH 8.3) for 1.25 hours at 400 V, and the gel was exposed on a Storage Phosphor Screen and imaged using a Typhoon PhosphoImager (GE Healthcare). Termination differences were quantified as described previously in Eckartt & Delbeau et al. 2024, comparing each reaction containing a Nus factor to its corresponding “no TF” reaction.
Institutions
- Rockefeller University