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Structural basis for substrate loading in bacterial RNA polymerase

Nature volume 448, pages 163–168 (2007)Cite this article

Abstract

The mechanism of substrate loading in multisubunit RNA polymerase is crucial for understanding the general principles of transcription yet remains hotly debated. Here we report the 3.0-Å resolution structures of the Thermus thermophilus elongation complex (EC) with a non-hydrolysable substrate analogue, adenosine-5′-[(α,β)-methyleno]-triphosphate (AMPcPP), and with AMPcPP plus the inhibitor streptolydigin. In the EC/AMPcPP structure, the substrate binds to the active (‘insertion’) site closed through refolding of the trigger loop (TL) into two α-helices. In contrast, the EC/AMPcPP/streptolydigin structure reveals an inactive (‘preinsertion’) substrate configuration stabilized by streptolydigin-induced displacement of the TL. Our structural and biochemical data suggest that refolding of the TL is vital for catalysis and have three main implications. First, despite differences in the details, the two-step preinsertion/insertion mechanism of substrate loading may be universal for all RNA polymerases. Second, freezing of the preinsertion state is an attractive target for the design of novel antibiotics. Last, the TL emerges as a prominent target whose refolding can be modulated by regulatory factors.

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Figure 1: Structures of the substrate complexes.
Figure 2: The insertion complex.
Figure 3: The preinsertion complex.
Figure 4: Effect of TL alterations on nucleotide addition rate.
Figure 5: Nucleotide addition cycle.

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Acknowledgements

We thank V. Svetlov for help with sequence alignments and critical reading of the manuscript. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Energy Research. This work was supported by grants from the NIH to D.G.V., I.A. and R.L.

Author Contributions D.G.V. determined and analysed the structure and supervised the project. M.N.V. performed crystallization and data collection. J.Z. and M.P. performed biochemical analysis of the trigger loop mutants under the guidance of R.L. I.A. performed biochemical analysis of the T. thermophilus TECs. D.G.V., I.A. and R.L. jointly wrote the manuscript.

The atomic coordinates and structure factors of the ttEC/AMPcPP and ttEC/AMPcPP/Stl complexes are deposited in the Protein Data Bank under accession numbers 2O5J and 2PPB, respectively.

Author information

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, Alabama 35294, USA,

    Dmitry G. Vassylyev & Marina N. Vassylyeva

  2. Department of Biomolecular Chemistry,,

    Jinwei Zhang

  3. Department of Biochemistry and,,

    Murali Palangat & Robert Landick

  4. Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706, USA,

    Robert Landick

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

    Irina Artsimovitch

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  1. Dmitry G. Vassylyev
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Correspondence to Dmitry G. Vassylyev.

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The atomic coordinates and structure factors of the ttEC/AMPcPP and ttEC/AMPcPP/Stl complexes are deposited in the Protein Data Bank under accession numbers 2O5J and 2PPB, respectively. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Discussion divided into two sections: ‘Pre-insertion state as an intermediate in the nucleotide addition cycle’ and ‘Comparison of the bacterial and eukaryotic substrate complexes’; Supplementary Tables 1-2 and Supplementary Figures 1-15 with Legends (PDF 2272 kb)

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Vassylyev, D., Vassylyeva, M., Zhang, J. et al. Structural basis for substrate loading in bacterial RNA polymerase. Nature 448, 163–168 (2007). https://doi.org/10.1038/nature05931

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