Pleomorphic effects of three small-molecule inhibitors on transcription elongation by <em>Mycobacterium tuberculosis</em> RNA polymerase

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 state..., 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., , # 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 po...,