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TALE proteins search DNA using a rotationally decoupled mechanism

Nature Chemical Biology volume 12, pages 831–837 (2016)Cite this article

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Abstract

Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins used extensively for gene editing. Despite recent progress, however, little is known about their sequence search mechanism. Here, we use single-molecule experiments to study TALE search along DNA. Our results show that TALEs utilize a rotationally decoupled mechanism for nonspecific search, despite remaining associated with DNA templates during the search process. Our results suggest that the protein helical structure enables TALEs to adopt a loosely wrapped conformation around DNA templates during nonspecific search, facilitating rapid one-dimensional (1D) diffusion under a range of solution conditions. Furthermore, this model is consistent with a previously reported two-state mechanism for TALE search that allows these proteins to overcome the search speed–stability paradox. Taken together, our results suggest that TALE search is unique among the broad class of sequence-specific DNA-binding proteins and supports efficient 1D search along DNA.

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Figure 1: Single-molecule imaging of TALEs during nonspecific search.
Figure 2: Effects of ionic strength on TALE diffusion.
Figure 3: Hydrodynamic flow assay for probing TALE diffusion along DNA.
Figure 4: Probe size dependence and mechanism of TALE diffusion along DNA.

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Acknowledgements

We thank T. Ha (University of Illinois Urbana–Champaign) for providing the plasmid for the aldehyde labeling scheme and S. Li for assistance in acquiring transmission electron microscope images of quantum dots. C.M.S. is funded by the David and Lucile Packard Foundation. H.Z. and L.C. are funded by the Institute for Genomic Biology at the University of Illinois at Urbana–Champaign. L.C. is funded by the FMC Corporation.

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Authors and Affiliations

  1. Department of Chemistry, University of Illinois at Urbana–Champaign, Urbana, Illinois, USA

    Luke Cuculis, Huimin Zhao & Charles M Schroeder

  2. Department of Biochemistry, University of Illinois at Urbana–Champaign, Urbana, Illinois, USA

    Zhanar Abil & Huimin Zhao

  3. Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana–Champaign, Urbana, Illinois, USA

    Huimin Zhao & Charles M Schroeder

  4. Center for Biophysics and Quantitative Biology, University of Illinois at Urbana–Champaign, Urbana, Illinois, USA

    Huimin Zhao & Charles M Schroeder

  5. Institute for Genomic Biology, University of Illinois at Urbana–Champaign, Urbana, Illinois, USA

    Huimin Zhao & Charles M Schroeder

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  1. Luke Cuculis
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Contributions

L.C., Z.A., H.Z., and C.M.S. designed experiments. Z.A. generated protein and DNA samples. L.C. performed single-molecule experiments and data analysis. L.C., Z.A., H.Z., and C.M.S. prepared the manuscript.

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Correspondence to Charles M Schroeder.

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The authors declare no competing financial interests.

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Cuculis, L., Abil, Z., Zhao, H. et al. TALE proteins search DNA using a rotationally decoupled mechanism. Nat Chem Biol 12, 831–837 (2016). https://doi.org/10.1038/nchembio.2152

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