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

Herrera-Asmat, O.; Tong, A. B.; Lin, W.; Kong, T.; Del Valle, J. R.; Guerra, D. G.; Ebright, Y. W.; Ebridght, R.; Bustamante, C.

doi:10.1101/2025.02.07.637008

bioRxiv · 10.1101/2025.02.07.637008

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

Herrera-Asmat, O.·Tong, A. B.·Lin, W.·Kong, T.·Del Valle, J. R.·Guerra, D. G.·Ebright, Y. W.·Ebridght, R.·Bustamante, C.

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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()-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.

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Herrera-Asmat, O., Tong, A. B., Lin, W., Kong, T., Del Valle, J. R., Guerra, D. G., Ebright, Y. W., Ebridght, R., Bustamante, C.. 2025-02-07. Pleomorphic effects of three small-molecule inhibitors on transcription elongation by Mycobacterium tuberculosis RNA polymerase. https://doi.org/10.1101/2025.02.07.637008

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