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Structural basis for transcription inhibition by tagetitoxin

Nature Structural & Molecular Biology volume 12, pages 1086–1093 (2005)Cite this article

Abstract

Tagetitoxin (Tgt) inhibits transcription by an unknown mechanism. A structure at a resolution of 2.4 Å of the Thermus thermophilus RNA polymerase (RNAP)–Tgt complex revealed that the Tgt-binding site within the RNAP secondary channel overlaps that of the stringent control effector ppGpp, which partially protects RNAP from Tgt inhibition. Tgt binding is mediated exclusively through polar interactions with the β and β′ residues whose substitutions confer resistance to Tgt in vitro. Importantly, a Tgt phosphate, together with two active site acidic residues, coordinates the third Mg2+ ion, which is distinct from the two catalytic metal ions. We show that Tgt inhibits all RNAP catalytic reactions and propose a mechanism in which the Tgt-bound Mg2+ ion has a key role in stabilization of an inactive transcription intermediate. Remodeling of the active site by metal ions could be a common theme in the regulation of catalysis by nucleic acid enzymes.

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Figure 1: RNAP–Tgt structure.
Figure 2: The Tgt-binding site.
Figure 3: Tgt and ppGpp bind to overlapping sites on RNAP.
Figure 4: Tgt inhibits all catalytic reactions.
Figure 5: Mechanism of Tgt action.

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Acknowledgements

We thank R. Landick and J. Roberts for helpful comments. This work was supported by grants GM74252 and GM74840 (to D.G.V.) and GM67153 (to I.A.) from the US National Institutes of Health, and by RIKEN (D.G.V.).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Schools of Medicine and Dentistry, 402B Kaul Genetics Building, 720 20th Street South, Birmingham, 35294, Alabama, USA

    Dmitry G Vassylyev, Marina N Vassylyeva & Anna Perederina

  2. Structural and Molecular Biology Laboratory, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, 679-5148, Hyogo, Japan

    Dmitry G Vassylyev, Marina N Vassylyeva & Anna Perederina

  3. Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, 43210, Ohio, USA

    Vladimir Svetlov & Irina Artsimovitch

  4. Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, 305-0801, Ibaraki, Japan

    Noriyuki Igarashi, Naohiro Matsugaki & Soichi Wakatsuki

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  1. Dmitry G Vassylyev
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Correspondence to Dmitry G Vassylyev or Irina Artsimovitch.

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Supplementary information

Supplementary Fig. 1

Tgt inhibits transcription by the E. coli and T. thermophilus RNAPs similarly (PDF 220 kb)

Supplementary Fig. 2

Comparison with the eukaryotic enzymes (PDF 120 kb)

Supplementary Fig. 3

Tgt acts as an uncompetitive inhibitor. (PDF 114 kb)

Supplementary Table 1

Plasmids and oligonucleotides used in this work (PDF 37 kb)

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Vassylyev, D., Svetlov, V., Vassylyeva, M. et al. Structural basis for transcription inhibition by tagetitoxin. Nat Struct Mol Biol 12, 1086–1093 (2005). https://doi.org/10.1038/nsmb1015

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