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Structural basis of transcription recognition of a hydrophobic unnatural base pair by T7 RNA polymerase

Author

Listed:
  • Juntaek Oh

    (University of California, San Diego)

  • Michiko Kimoto

    (Agency for Science, Technology and Research (A*STAR)
    Xenolis Pte. Ltd.)

  • Haoqing Xu

    (University of California, San Diego)

  • Jenny Chong

    (University of California, San Diego)

  • Ichiro Hirao

    (Agency for Science, Technology and Research (A*STAR)
    Xenolis Pte. Ltd.)

  • Dong Wang

    (University of California, San Diego
    University of California, San Diego
    University of California, San Diego)

Abstract

Bacteriophage T7 RNA polymerase (T7 RNAP) is widely used for synthesizing RNA molecules with synthetic modifications and unnatural base pairs (UBPs) for a variety of biotechnical and therapeutic applications. However, the molecular basis of transcription recognition of UBPs by T7 RNAP remains poorly understood. Here we focused on a representative UBP, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole 2-carbaldehyde (Pa), and investigated how the hydrophobic Ds–Pa pair is recognized by T7 RNAP. Our kinetic assays revealed that T7 RNAP selectively recognizes the Ds or Pa base in the templates and preferentially incorporates their cognate unnatural base nucleotide substrate (PaTP or DsTP) over natural NTPs. Our structural studies reveal that T7 RNAP recognizes the unnatural substrates at the pre-insertion state in a distinct manner compared to natural substrates. These results provide mechanistic insights into transcription recognition of UBP by T7 RNAP and provide valuable information for designing the next generation of UBPs.

Suggested Citation

  • Juntaek Oh & Michiko Kimoto & Haoqing Xu & Jenny Chong & Ichiro Hirao & Dong Wang, 2023. "Structural basis of transcription recognition of a hydrophobic unnatural base pair by T7 RNA polymerase," Nature Communications, Nature, vol. 14(1), pages 1-8, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-022-35755-8
    DOI: 10.1038/s41467-022-35755-8
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    References listed on IDEAS

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    1. Dmitry G. Vassylyev & Marina N. Vassylyeva & Jinwei Zhang & Murali Palangat & Irina Artsimovitch & Robert Landick, 2007. "Structural basis for substrate loading in bacterial RNA polymerase," Nature, Nature, vol. 448(7150), pages 163-168, July.
    2. Tahir H. Tahirov & Dmitry Temiakov & Michael Anikin & Vsevolod Patlan & William T. McAllister & Dmitry G. Vassylyev & Shigeyuki Yokoyama, 2002. "Structure of a T7 RNA polymerase elongation complex at 2.9 Å resolution," Nature, Nature, vol. 420(6911), pages 43-50, November.
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    Cited by:

    1. Juntaek Oh & Zelin Shan & Shuichi Hoshika & Jun Xu & Jenny Chong & Steven A. Benner & Dmitry Lyumkis & Dong Wang, 2023. "A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase," Nature Communications, Nature, vol. 14(1), pages 1-10, December.
    2. Karl Herbine & Ashok R. Nayak & Dmitry Temiakov, 2024. "Structural basis for substrate binding and selection by human mitochondrial RNA polymerase," Nature Communications, Nature, vol. 15(1), pages 1-9, December.

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