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Structural basis of HutP-mediated anti-termination and roles of the Mg2+ ion and L-histidine ligand

Nature volume 434, pages 183–191 (2005)Cite this article

Abstract

HutP regulates the expression of the hut structural genes of Bacillus subtilis by an anti-termination mechanism and requires two components, Mg2+ ions and l-histidine. HutP recognizes three UAG triplet units, separated by four non-conserved nucleotides on the terminator region. Here we report the 1.60-Å resolution crystal structure of the quaternary complex (HutP–l-histidine–Mg2+–21-base single-stranded RNA). In the complex, the RNA adopts a novel triangular fold on the hexameric surface of HutP, without any base-pairing, and binds to the protein mostly by specific protein–base interactions. The structure explains how the HutP and RNA interactions are regulated critically by the l-histidine and Mg2+ ion through the structural rearrangement. To gain insights into these structural rearrangements, we solved two additional crystal structures (uncomplexed HutP and HutP–l-histidine–Mg2+) that revealed the intermediate structures of HutP (before forming an active structure) and the importance of the Mg2+ ion interactions in the complexes.

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Figure 1: Terminator structure of hut mRNA, and the overall structure of the HutP quaternary complex.
Figure 2: HutP–RNA interactions.
Figure 3: l-histidine and Mg2+ interactions in the HutP quaternary complex.
Figure 4: Stereo views of conformational changes observed in the quaternary complex.
Figure 5: Electrostatic surface potential models of HutP and the proposed mechanism for the anti-terminator complex formation.

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Acknowledgements

We thank the personnel at beamline AR-NW12 of the Photon Factory, Tsukuba, Japan for assistance during data collection, and T. Misono for help in the purification of HutP mutants and in sequence analysis. T.S.K. holds a fellowship of the AIST, Tsukuba, Japan. This work was supported by funds from METI and the ORCS project to P.K.R.K. and H.M., respectively.

Author information

Author notes

  1. Hiroshi Mizuno

    Present address: NEC Soft. Ltd, 1-18-6, Shinkiba, Koto-ku, Tokyo, 106-8608, Japan

Authors and Affiliations

  1. Functional Nucleic Acids Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan

    Thirumananseri Kumarevel & Penmetcha K. R. Kumar

  2. Department of Biochemistry, National Institute of Agrobiological Sciences, 2-1-2, Kannondai, Tsukuba, 305-8602, Ibaraki, Japan

    Hiroshi Mizuno

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Corresponding author

Correspondence to Penmetcha K. R. Kumar.

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Supplementary information

Supplementary Table S1

This table contains the data collection and refinement statistics of the three crystal structures presented in this manuscript. (DOC 46 kb)

Supplementary Discussion 1

This file contains the comparison analyses of the RNA conformations from the HutP and TRAP protein. (DOC 21 kb)

Supplementary Figure S1

This provides details of the interactions of HutP with the L-histidine analogue. (JPG 35 kb)

Supplementary Figure S2

Mutational analysis of the RNA binding site using a filter binding assay. (JPG 36 kb)

Supplementary Figure S3

Superposition of the important regions of the RNA structures in the HutP and TRAP proteins. (JPG 81 kb)

Supplementary Figure S4

The conserved important amino acid residues in the HutP protein among various Bacillus species. (JPG 113 kb)

Supplementary Figure S5

Conformational changes observed in the ternary complex. (JPG 102 kb)

Supplementary Figure S6

Superposition of the HutP crystal structures, shown in the front (a) and back (b) side views. (JPG 239 kb)

Supplementary Figure Legends

This file contains the legends for the above Supplementary Figures S1-S6. (DOC 23 kb)

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Kumarevel, T., Mizuno, H. & Kumar, P. Structural basis of HutP-mediated anti-termination and roles of the Mg2+ ion and L-histidine ligand. Nature 434, 183–191 (2005). https://doi.org/10.1038/nature03355

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