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The 2′-OH group at the group II intron terminus acts as a proton shuttle

Nature Chemical Biology volume 6, pages 218–224 (2010)Cite this article

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Abstract

Group II introns are self-splicing ribozymes that excise themselves from precursor RNAs and catalyze the joining of flanking exons. Excised introns can behave as parasitic RNA molecules: they can catalyze their own insertion into DNA and RNA via a reverse splicing reaction. Previous studies have identified mechanistic roles for various functional groups located in the catalytic core of the intron and within target molecules. Here we introduce a new method for synthesizing long RNA molecules with a modified nucleotide at the 3′ terminus. This modification allows us to examine the mechanistic role of functional groups adjacent to the reaction nucleophile. During reverse splicing, the 3′-OH group of the intron terminus attacks the phosphodiester linkage of spliced exon sequences. Here we show that the adjacent 2′-OH group on the intron terminus plays an essential role in activating the nucleophile by stripping away a proton from the 3′-OH and then shuttling it from the active site.

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Figure 1: Structure and splicing pathways of group II introns.
Figure 2: Synthesis and verification of intron RNAs with substitutions of the 2′-OH group at the 3′-terminal nucleotide.
Figure 3: Reverse splicing assay.
Figure 4: Suggested proton shuttle mechanism.

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Acknowledgements

We thank C.M. Joyce (Yale University) for providing the E710A mutant protein. This work was supported by generous funding from the US National Institutes of Health (GM50313) and from the Howard Hughes Medical Institute. A.M.P. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Howard Hughes Medical Institute, Yale University, New Haven, Connecticut, USA

    Michael Roitzsch, Olga Fedorova & Anna Marie Pyle

  2. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Michael Roitzsch, Olga Fedorova & Anna Marie Pyle

  3. Fakultät Chemie, Technische Universität Dortmund, Dortmund, Germany

    Michael Roitzsch

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Contributions

M.R. designed and performed kinetic experiments and analyzed the data. M.R. designed and conducted the method for generation of 3′-terminally modified RNAs including their purity control, but with the exception of experiments involving fluorescein labeling. O.F. designed and performed all control experiments involving fluorescein labeling and prepared the modified DNA primers and all synthetic RNA oligonucleotides. M.R. interpreted the results and wrote the manuscript. O.F. helped interpret the results. O.F. and A.M.P. edited the manuscript. A.M.P provided funding for the research and supervised all experimentation.

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Correspondence to Anna Marie Pyle.

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Roitzsch, M., Fedorova, O. & Pyle, A. The 2′-OH group at the group II intron terminus acts as a proton shuttle. Nat Chem Biol 6, 218–224 (2010). https://doi.org/10.1038/nchembio.312

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