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Structural and biochemical studies of the 5′→3′ exoribonuclease Xrn1

Nature Structural & Molecular Biology volume 18, pages 270–276 (2011)Cite this article

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Abstract

The 5′→3′ exoribonucleases (XRNs) have important functions in transcription, RNA metabolism and RNA interference. The structure of Rat1 (also known as Xrn2) showed that the two highly conserved regions of XRNs form a single, large domain that defines the active site of the enzyme. Xrn1 has a 510-residue segment after the conserved regions that is required for activity but is absent from Rat1/Xrn2. Here we report the crystal structures of Kluyveromyces lactis Xrn1 (residues 1–1,245, E178Q mutant), alone and in complex with a Mn2+ ion in the active site. The 510-residue segment contains four domains (D1–D4), located far from the active site. Our mutagenesis and biochemical studies show that their functional importance results from their ability to stabilize the conformation of the N-terminal segment of Xrn1. These domains might also constitute a platform that interacts with protein partners of Xrn1.

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Figure 1: Domain organization of Xrn1.
Figure 2: Structure of K. lactis Xrn1.
Figure 3: Unique structural features in Xrn1.
Figure 4: Structure of the active site of K. lactis Xrn1.
Figure 5: Biochemical studies on the nuclease activity of K. lactis Xrn1.
Figure 6: RNA binding activity of K. lactis Xrn1.
Figure 7: Conserved surface features in the structure of K. lactis Xrn1.

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Acknowledgements

We thank N. Whalen and S. Myers for setting up the X29A beamline at the National Synchrontron Light Source (NSLS). This research was supported in part by grants from the US National Institutes of Health to L.T. (GM090059 and GM077175) and J.L.M. (GM028983).

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Author notes

  1. Song Xiang

    Present address: Present address: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, P.R. China.,

  2. Jeong Ho Chang and Song Xiang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Columbia University, New York, New York, USA

    Jeong Ho Chang, Song Xiang, Kehui Xiang, James L Manley & Liang Tong

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  1. Jeong Ho Chang
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  2. Song Xiang
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Contributions

J.H.C. and S.X. carried out protein expression, purification and crystallization experiments. J.H.C., S.X. and L.T. carried out crystallographic data collection, structure determination and refinement. J.H.C. carried out mutagenesis, nuclease assays, EMSA and thermal shift experiments. K.X. helped with the preparation of the nuclease substrate. J.L.M. designed experiments and analyzed data. All authors commented on the manuscript. L.T. designed experiments, analyzed data and wrote the paper.

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Correspondence to Liang Tong.

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Chang, J., Xiang, S., Xiang, K. et al. Structural and biochemical studies of the 5′→3′ exoribonuclease Xrn1. Nat Struct Mol Biol 18, 270–276 (2011). https://doi.org/10.1038/nsmb.1984

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