Pleomorphic effects of three small-molecule inhibitors on transcription elongation by Mycobacterium tuberculosis RNA polymerase
- University of California, Berkeley, United States
- Shenzhen University, China
- University of Notre Dame, United States
- Universidad Peruana Cayetano Heredia, Peru
- Rutgers, The State University of New Jersey, United States
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.
Data availability
All oligos/plasmids used in this study are available upon reasonable request. See Supplementary Tables S1-S3 for a list of materials.Transcription traces are available from Dryad at https://doi.org/10.5061/dryad.2fqz6130m.Matlab code used for analysis is available on Github at https://doi.org/10.5281/zenodo.15232932.
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Single-molecule optical tweezers data of transcription of M.tuberculosis and E.coli RNAP in the presence of single-molecule inhibitorsDryad Digital Repository, doi:10.5061/dryad.2fqz6130m.
Article and author information
Author details
Funding
National Institutes of Health (GM041376)
- Richard H Ebridght
National Institutes of Health (R01GM032543)
- Carlos Bustamante
National Natural Science Foundation of China (31900883)
- Wenxia Lin
Howard Hughes Medical Institute
- Carlos Bustamante
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2025, Herrera-Asmat et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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Pleomorphic effects of three small-molecule inhibitors on transcription elongation by Mycobacterium tuberculosis RNA polymerase
eLife 14:e105545.
https://doi.org/10.7554/eLife.105545
