DOI: 10.5061/dryad.2fqz6130m
title: Data from: Pleomorphic effects of three small-molecule inhibitors on transcription elongation by <em>Mycobacterium tuberculosis</em> RNA polymerase

description: 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.
description: 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.
description: # Data from: Pleomorphic effects of three small-molecule inhibitors on
 transcription elongation by *Mycobacterium tuberculosis* RNA polymerase
 [https://doi.org/10.5061/dryad.2fqz6130m](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](https://github.com/abmtong/BLabOTMatlab). See the readme for this project there for specifics on what files were used.