Data from: Pleomorphic effects of three small-molecule inhibitors on transcription elongation by Mycobacterium tuberculosis RNA polymerase
Data files
Oct 29, 2025 version files 6.53 GB
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Dryad_Summary_of_Traces.xlsx
13.45 KB
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Herrera-Asmat_et_al_Transcription_Traces_V2.zip
6.53 GB
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README.md
4.58 KB
Abstract
The Mycobacterium tuberculosis RNA polymerase (MtbRNAP) is the target of the first-line anti-tuberculosis inhibitor rifampin, however, the emergence of rifampin resistance necessitates the development of new antibiotics. Here, we communicate the first single-molecule characterization of MtbRNAP elongation and its inhibition by three diverse small-molecule inhibitors: N(α)-aroyl-N-aryl-phenylalaninamide (D-IX216), streptolydigin (Stl), and pseudouridimycin (PUM) using high-resolution optical tweezers. Compared to Escherichia coli RNA polymerase (EcoRNAP), MtbRNAP transcribes more slowly, has similar mechanical robustness, and only weakly recognizes E. coli pause sequences. The three small-molecule inhibitors of MtbRNAP exhibit strikingly different effects on transcription elongation. In the presence of D-IX216, which inhibits RNAP active-center bridge-helix motions required for nucleotide addition, the enzyme exhibits transitions between slowly and super-slowly elongating inhibited states. Stl, which inhibits the RNAP trigger-loop motions also required for nucleotide addition, inhibits RNAP primarily by inducing pausing and backtracking. PUM, a nucleoside analog of UTP, in addition to acting as a competitive inhibitor, induces the formation of slowly elongating RNAP inhibited states. Our results indicate that the three classes of small-molecule inhibitors affect the enzyme in distinct ways and show that the combination of Stl and D-IX216, which both target the RNAP bridge helix, has a strong synergistic effect on the enzyme.
https://doi.org/10.5061/dryad.2fqz6130m
Description of the data and file structure
Enclosed are single-molecule optical tweezers data for transcription of E.coli or M. tuberculosis RNA polymerase.
They are separated by experiment type and condition.
Folders:
Assisting or Opposing Force Mode: Experiments done in force feedback mode, where the force is held constant. Experiments where transcription causes the tether length to increase are labeled 'Assisting force mode', since the force of the traps acts to assist the polymerase. Experiments where transcription causes the tether length to decrease are labeled 'Opposing force mode', since the force of the traps acts to hinder the polymerase's motion.
Passive Mode: Experiments done in passive mode, where the optical traps are held at constant position, causing force to rise as transcription progresses.
See the subfolders for more information about the exact experiments and where they show up in the paper.
Semipassive Mode: Experiments done in semipassive assisting mode, where the optical traps are moved stepwise to keep the force within a set range. In other words, the traps are held at constant position, causing the force to fall as transcription progresses, until it reaches some lower force limit (let's say 10pN) after which the traps move to raise the force to some upper force limit (say 18pN). Thus, transcription occurs within a range of forces, in this case 10-18pN.
Files and variables
All traces are zipped in Herrera-Asmat_et_al_Transcription_Traces_V2.zip. Inside are folders containing the experimental conditions, more details in Dryad_Summary_of_Traces.xlsx. Some experimental conditions contain subfolders such as "CropFiles", which are text files containing the region of data analyzed, and "Broken", which have traces that were not analyzed; these files can be ignored.
Data are saved as a MATLAB .mat file, one per trace. Inside is a Matlab structure with fields:
- forceAX/BX/AY/BY: Individual measured forces in each of the two traps A and B in the X and Y directions. The experiment happens along the X direction. Unit: piconewton (pN).
- extension: The extension (end-to-end distance) of the tether. Unit: nanometer (nm).
- force: The net force applied on the tether, taken as the (pythagorean) sum of the X and Y forces.
- time: Time (seconds).
- contour: The contour length (length of DNA in the tether, in basepairs) of the tether, the conversion from extension to contour is modeled via the extensible worm-like chain model for DNA.
The other fields are either unused or relate to calibration or settings.
The file Dryad_Summary_of_Traces.xlsx has these columns:
- RNAP: The organism (Eco = Ecoli or Mtb = Mycobacterium Tuberculosis) the RNAP is from in the experiment
- Template: The DNA template for transcription used in the experiment.
- Experimental Mode: The mode that the experiment is done in: This is either Assisting/Opposing Constant Force, where the instrument keeps the force applied to the tether constant via moving the traps by feedback, or Assisting Semipassive Mode, where the instrument moves the traps in large steps to release the tension after the force rises above a set amount. Semipassive involves two forces, the upper limit (after which the instrument moves the traps), and the lower limit (the force of the tether after each trap movement).
- Applied Force (pN, + is assisting, - is opposing): The magnitude and direction of the applied force. Assisting is designated as positive and opposing is negative forces. For semipassive, as there are two forces involved, it is stated as a range of low to high force.
- rNTP Condition: The rNTP concentration used for this experiment. If only one concentration is stated, then it is equal concentration of all four rNTPs.
- Inhibitor/ Factor?: The protein or small molecule added to the transcription medium and its concentration.
- Used in Figure?: The figure that the data corresponds to in the eLife paper.
- N Traces: The number of traces in the folder.
Dryad Folder Name: The full path to the folder with the traces.
Code/software
Custom code used for visualization and analysis are written in Matlab are available at https://github.com/abmtong/BLabOTMatlab. See the readme for this project there for specifics on what files were used.
Data were collected with a dual-trap timeshared optical tweezers system, similar to one described in doi:10.1038/NMETH.1574, except without fluorescence capability. A single RNAP molecule was tethered between two beads caught in the two traps via DNA handles, and as the RNAP transcribed along the DNA, the amount of DNA that made up the tether would change, which would be sensed in the instrument as a change in force. Depending on the type of experiment performed, either the force was allowed to rise ('passive mode') or the separation of the traps was adjusted to keep the force constant ('force feedback mode')
Raw instrument data (QPD voltages, AOD frequency) were processed into force (pN) and extension (nm) data using a calibration to the power spectrum of beads caught in the traps without a tether. The extension of the tether was converted into contour length using the extensible worm-like chain model. These processed values were saved in Matlab format.