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


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.


  1. Chasin, L. A. & Magasanik, B. Induction and repression of the histidine-degrading enzymes of Bacillus subtilis. J. Biol. Chem. 243, 5165–5178 (1968)

    CAS  PubMed  Google Scholar 

  2. Kimmhi, Y. & Magasanik, B. Genetic basis of histidine degradation in Bacillus subtilis. J. Biol. Chem. 245, 3545–3548 (1970)

    Google Scholar 

  3. Oda, M., Sugishita, A. & Furukawa, K. Cloning and nucleotide sequences of histidase and regulatory genes in the Bacillus subtilis hut operon and positive regulation of the operon. J. Bacteriol. 170, 3199–3205 (1988)

    Article  CAS  Google Scholar 

  4. Wray, L. V. Jr & Fisher, S. H. Analysis of Bacillus subtilis hut operon expression indicates that histidine-dependent induction is mediated primarily by transcriptional anti-termination and that amino acid repression is mediated by two mechanisms: regulation of transcription initiation and inhibition of histidine transport. J. Bacteriol. 176, 5466–5473 (1994)

    Article  CAS  Google Scholar 

  5. Oda, M., Kobayashi, N., Ito, A., Kurusu, Y. & Taira, K. Cis-acting regulatory sequences for anti-termination in the transcript of the Bacillus subtilis hut operon and histidine-dependent binding of HutP to the transcript containing the regulatory sequences. Mol. Microbiol. 35, 1244–1254 (2000)

    Article  CAS  Google Scholar 

  6. Yanofsky, C. Advancing our knowledge in biochemistry, genetics, and microbiology through studies on tryptophan metabolism. Annu. Rev. Biochem. 70, 1–37 (2001)

    Article  CAS  Google Scholar 

  7. Gollnick, P. & Babitzke, P. Transcription attenuation. Biochim. Biophys. Acta 1577, 240–245 (2002)

    Article  CAS  Google Scholar 

  8. Stulke, J. Control of transcription termination in bacteria by RNA-binding proteins that modulate RNA structures. Arch. Microbiol. 177, 433–440 (2002)

    Article  CAS  Google Scholar 

  9. Houman, F., Diaz-Torres, M. R. & Wright, A. Transcriptional anti-termination in the bgl operon of E. coli is modulated by a specific RNA binding protein. Cell 62, 1153–1163 (1990)

    Article  CAS  Google Scholar 

  10. Aymerich, S. & Stenmetz, M. Specificity determinants and structural features in the RNA target of the bacterial anti-terminator proteins of the BgiG/SacY family. Proc. Natl Acad. Sci. USA 89, 10410–10414 (1992)

    Article  ADS  CAS  Google Scholar 

  11. Babitzke, P. & Yanofsky, C. Reconstitution of Bacillus subtilis trp attenuation in vitro with TRAP, the trp RNA-binding attenuation protein. Proc. Natl Acad. Sci. USA 90, 133–137 (1993)

    Article  ADS  CAS  Google Scholar 

  12. Arnaud, M. D., Debarbouille, M., Rapoport, G., Saier, M. H. Jr & Reizer, J. In vitro reconstitution of transcriptional attenuation by the SacT and SacY proteins of Bacillus subtilis. J. Biol. Chem. 271, 18966–18972 (1996)

    Article  CAS  Google Scholar 

  13. Lu, Y., Turner, R. J. & Switzer, R. L. Function of RNA secondary structures in transcriptional attenuation of the Bacillus subtilis pyr operon. Proc. Natl Acad. Sci. USA 93, 14462–14467 (1996)

    Article  ADS  CAS  Google Scholar 

  14. Alpert, C. A. & Siebers, U. The lac operon of Lactobacillus casei contains lacT, a gene coding for a protein of the Bg1G family of transcriptional anti-terminators. J. Bacteriol. 179, 1555–1562 (1997)

    Article  CAS  Google Scholar 

  15. Glatz, E., Nilsson, R. P., Rutberg, L. & Rutberg, B. A dual role for the Bacillus subtilis glpD leader and the GlpP protein in the regulated expression of glpD: anti-termination and control of mRNA stability. Mol. Microbiol. 19, 319–328 (1996)

    Article  CAS  Google Scholar 

  16. Kumarevel, T. S. et al. Crystal structure of activated HutP: an RNA binding protein that regulates hut operon in Bacillus subtilis. Structure 12, 1269–1280 (2004)

    Article  CAS  Google Scholar 

  17. Kumarevel, T. S., Mizuno, H. & Kumar, P. K. R. Allosteric activation of HutP protein, that regulates transcription of hut operon in Bacillus subtilis, mediated by various analogs of histidine. Nucleic Acids Res. Suppl. 3, 199–200 (2003)

    Article  CAS  Google Scholar 

  18. Kumarevel, T. S., Gopinath, S. C. B., Mizuno, H. & Kumar, P. K. R. Identification of important chemical groups of the hut mRNA for HutP interactions that regulates the hut operon in Bacillus subtilis. Nucleic Acids Res. 32, 3904–3912 (2004)

    Article  CAS  Google Scholar 

  19. Read, T. D. et al. The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature 423, 81–86 (2003)

    Article  ADS  CAS  Google Scholar 

  20. Ivanova, N. et al. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423, 87–91 (2003)

    Article  ADS  CAS  Google Scholar 

  21. Takami, H. et al. Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res. 28, 4317–4331 (2000)

    Article  CAS  Google Scholar 

  22. Antson, A. A. et al. Structure of the trp RNA-binding attenuation protein, TRAP, bound to RNA. Nature 401, 235–242 (1999)

    Article  ADS  CAS  Google Scholar 

  23. Yang, Y., Declerck, N., Manivel, X., Aymerich, S. & Kochoyan, M. Solution structure of the LicT-RNA anti-termination complex: CAT clamping RAT. EMBO J. 21, 1987–1997 (2002)

    Article  CAS  Google Scholar 

  24. Wimberly, B. T., Guymon, R., McCutcheon, J. P., White, S. W. & Ramakrishnan, V. A detailed view of a ribosomal active site: The structure of the L11–RNA complex. Cell 97, 491–502 (1999)

    Article  CAS  Google Scholar 

  25. Deo, R. C., Bonanno, J. B., Sonenberg, N. & Burely, S. K. Recognition of polyadenylate RNA by the poly(A)-binding protein. Cell 98, 835–845 (1999)

    Article  CAS  Google Scholar 

  26. Handa, N. et al. Structural basis for recognition of the tra mRNA precursor by the Sex-lethal protein. Nature 398, 579–585 (1999)

    Article  ADS  CAS  Google Scholar 

  27. Liu, Z. et al. Structural basis for recognition of the intron branch site RNA by splicing factor 1. Science 294, 1098–1102 (2001)

    Article  ADS  CAS  Google Scholar 

  28. Lewis, H. A. et al. Sequence-specific RNA binding by a Nova KH domain: implications for paraneoplastic disease and fragile X syndrome. Cell 100, 323–332 (2000)

    Article  CAS  Google Scholar 

  29. Bogden, C. E., Fass, D., Bergman, N., Nichols, M. D. & Berger, J. M. Structural basis for terminator recognition by the Rho transcription termination factor. Mol. Cell 3, 487–493 (1999)

    Article  CAS  Google Scholar 

  30. Thore, S., Mayes, C., Sauter, C., Weeks, S. & Suck, D. Crystal structures of the Pyrococcus abyssi Sm core and its complex with RNA. Common features of RNA binding in Archaea and Eukarya. J. Biol. Chem. 278, 1239–1247 (2003)

    Article  CAS  Google Scholar 

  31. Nagai, K., Outbridge, C., Jessen, T. H., Li, J. & Evans, P. R. Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A. Nature 348, 515–520 (1990)

    Article  ADS  CAS  Google Scholar 

  32. Kumarevel, T. S. et al. Crystallization and preliminary X-ray diffraction studies of HutP protein: an RNA binding protein that regulates the transcription of hut operon in Bacillus subtilis. J. Struct. Biol. 138, 237–240 (2002)

    Article  CAS  Google Scholar 

  33. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation model. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  34. Brunger, A. et al. Crystallography and NMR system: a new software system for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  CAS  Google Scholar 

  35. Oldfield, T. J. A number of real-space torsion-angle refinement techniques for proteins, nucleic acids, ligands and solvent. Acta Crystallogr. D 57, 82–94 (2001)

    Article  CAS  Google Scholar 

  36. Carson, M. Ribbons. Methods Enzymol. 277, 493–505 (1997)

    Article  CAS  Google Scholar 

  37. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991)

    Article  CAS  Google Scholar 

  38. Kraulis, P. J. Molscript: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991)

    Article  Google Scholar 

  39. Merritt, E. A. & Bacon, D. J. Raster3D photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997)

    Article  CAS  Google Scholar 

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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.

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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).

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