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Structural basis for RNA trimming by RNase T in stable RNA 3′-end maturation

Nature Chemical Biology volume 7, pages 236–243 (2011)Cite this article

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Abstract

RNA maturation relies on various exonucleases to remove nucleotides successively from the 5′ or 3′ end of nucleic acids. However, little is known regarding the molecular basis for substrate and cleavage preference of exonucleases. Our biochemical and structural analyses on RNase T–DNA complexes show that the RNase T dimer has an ideal architecture for binding a duplex with a short 3′ overhang to produce a digestion product of a duplex with a 2-nucleotide (nt) or 1-nt 3′ overhang, depending on the composition of the last base pair in the duplex. A 'C-filter' in RNase T screens out the nucleic acids with 3′-terminal cytosines for hydrolysis by inducing a disruptive conformational change at the active site. Our results reveal the general principles and the working mechanism for the final trimming step made by RNase T in the maturation of stable RNA and pave the way for the understanding of other DEDD family exonucleases.

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Figure 1: The crystal structure of the preferred RNase T–DNA complex.
Figure 2: The crystal structure of the nonpreferred RNase T–DNA complexes.
Figure 3: A C-filter in RNase T deselects DNA with a 3′-terminal C for cleavage.
Figure 4: Crystal structure of RNase T in complex with a duplex DNA with a 2-nt 3′ overhang.
Figure 5: RNase T cleaves a 3′ overhang duplex to produce a duplex with a 1- or 2-nt 3′ overhang.
Figure 6: General principles of the 3′-terminal final trimming made by RNase T in RNA maturation.

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Acknowledgements

This work was supported by research grants from Academia Sinica and the National Science Council, Taiwan. Portions of this research were carried out at the National Synchrotron Radiation Research Center (BL-13B1 and BL-13C1), a national user facility supported by the National Science Council of Taiwan. The Synchrotron Radiation Protein Crystallography Facility is supported by the National Research Program for Genomic Medicine. We thank S. Lin-Chao (Academia Sinica) for providing the E. coli knockout strains used in this study.

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Authors and Affiliations

  1. Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan, ROC

    Yu-Yuan Hsiao & Chia Liang Lin

  2. Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC

    Yu-Yuan Hsiao, Che-Chuan Yang, Chia Liang Lin, Jason L J Lin, Yulander Duh & Hanna S Yuan

  3. Graduate Institute of Biochemistry and Molecular Biology, National Taiwan University, Taipei, Taiwan, ROC

    Che-Chuan Yang & Hanna S Yuan

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Contributions

Y.-Y.H. and H.S.Y. designed experiments and analyzed data. Y.-Y.H., C.-C.Y., C.L.L., J.L.J.L. and Y.D. carried out biochemical and structural analysis.

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Correspondence to Hanna S Yuan.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–3 (PDF 4579 kb)

Supplementary Movie 1

The crystal structure of RNase T bound to a preferred ssDNA with a 3′-end G. The ssDNA is bound at a deep narrow groove near the DEDD active site (PDB ID: 3NGY and 3NH1). Four aromatic residues make π-π stacking interactions with the two 3′-terminal bases. Two Mg2+ ions are bound at the DEDD active site in an active conformation. (MOV 8750 kb)

Supplementary Movie 2

RNase T bound to a non-preferred ssDNA with a 3′-end C induces disruptive conformational changes at the active site. The side chain of Glu73 is rotated to make H-bonds with the 3′-terminal C and the aromatic side chains of Phe29 and Phe27 are shifted to new positions to stack with the 3′-terminal C in the RNase T–DNA2 complex (PDB ID: 3NGZ). As a result, the scissile phosphate in the nonpreferred complex moves up away from the active site and only one Mg2+ ion is bound at the active site in an inactive conformation. (MOV 12732 kb)

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Hsiao, YY., Yang, CC., Lin, C. et al. Structural basis for RNA trimming by RNase T in stable RNA 3′-end maturation. Nat Chem Biol 7, 236–243 (2011). https://doi.org/10.1038/nchembio.524

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