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A single active-site region for a group II intron

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Abstract

Despite the biological importance of self-splicing group II introns, little is known about their structural organization. Synthetic incorporation of site-specific photo-cross-linkers within catalytic domains resulted in functional distance constraints that, when combined with known tertiary interactions, provide a three-dimensional view of the active intron architecture. All functionalities important for both steps of splicing are proximal before the first step, suggestive of a single active-site region for group II intron catalysis.

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Figure 1: Photo-cross-links from the branch site and from the 3′ exon.
Figure 2: A close-up view of the active-site region, derived from the ai5γ group II intron model (Supplementary Fig. 4 online).

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Acknowledgements

We thank O. Fedorova for help with photo-cross-linking and RNA synthesis, L. Wadley, M. Schmeing and J. Wang for help with modeling and L. Shapiro for helpful discussions. This research was supported by fellowships from Praxis XXI, Gulbenkian 43058 (A.D.L.) and by US National Institutes of Health grants GM0822414, and GM0822415 (S.H.) and GM50313 (A.M.P.). A.M.P. is an Investigator with the Howard Hughes Medical Institute.

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Author notes

  1. Alexandre de Lencastre and Stephanie Hamill: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Columbia University, 630 West 168th St., New York, 10032, New York, USA

    Alexandre de Lencastre

  2. Integrated Program in Cellular, Molecular and Biophysical Studies, Columbia University, 630 West 168th St., New York, 10032, New York, USA

    Stephanie Hamill

  3. Department of Molecular Biophysics and Biochemistry, Yale University, Room 334A, 266 Whitney Avenue, New Haven, 06520, Connecticut, USA

    Anna Marie Pyle

  4. Howard Hughes Medical Institute, Yale University, Room 334A, 266 Whitney Avenue, New Haven, 06520, Connecticut, USA

    Anna Marie Pyle

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  1. Alexandre de Lencastre
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  2. Stephanie Hamill
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  3. Anna Marie Pyle
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Corresponding author

Correspondence to Anna Marie Pyle.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Schematic group II intron secondary structure. (PDF 412 kb)

Supplementary Fig. 2

The products of splicing from a D56 molecule containing a single-nucleotide 3′-exon. (PDF 410 kb)

Supplementary Fig. 3

Secondary structure of the intron with color coding of corresponding domains in the tertiary structure that were modeled (Supplementary Fig. 4). (PDF 957 kb)

Supplementary Fig. 4

Three-dimensional model, color-coded as in Supplementary Fig. 3. (PDF 198 kb)

Supplementary Table 1

Crosslinks identified in the present study. (PDF 67 kb)

Supplementary Table 2

Additional known constraints on group II intron architecture. (PDF 101 kb)

Supplementary Methods (PDF 93 kb)

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de Lencastre, A., Hamill, S. & Pyle, A. A single active-site region for a group II intron. Nat Struct Mol Biol 12, 626–627 (2005). https://doi.org/10.1038/nsmb957

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