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. 1998 Sep 15;17(18):5449–5457. doi: 10.1093/emboj/17.18.5449

Functional analysis of peptide motif for RNA microhelix binding suggests new family of RNA-binding domains.

L Ribas de Pouplana 1, D Buechter 1, N Y Sardesai 1, P Schimmel 1
PMCID: PMC1170870  PMID: 9736622

Abstract

RNA microhelices that recreate the acceptor stems of transfer RNAs are charged with specific amino acids. Here we identify a two-helix pair in alanyl-tRNA synthetase that is required for RNA microhelix binding. A single point mutation at an absolutely conserved residue in this motif selectively disrupts RNA binding without perturbation of the catalytic site. These results, and findings of similar motifs in the proximity of the active sites of other tRNA synthetases, suggest that two-helix pairs are widespread and provide a structural framework important for contacts with bound RNA substrates.

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Selected References

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  1. Aberg A., Yaremchuk A., Tukalo M., Rasmussen B., Cusack S. Crystal structure analysis of the activation of histidine by Thermus thermophilus histidyl-tRNA synthetase. Biochemistry. 1997 Mar 18;36(11):3084–3094. doi: 10.1021/bi9618373. [DOI] [PubMed] [Google Scholar]
  2. Alexandrov N. N., Fischer D. Analysis of topological and nontopological structural similarities in the PDB: new examples with old structures. Proteins. 1996 Jul;25(3):354–365. doi: 10.1002/(SICI)1097-0134(199607)25:3<354::AID-PROT7>3.0.CO;2-F. [DOI] [PubMed] [Google Scholar]
  3. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  4. Arnez J. G., Cavarelli J. Structures of RNA-binding proteins. Q Rev Biophys. 1997 Aug;30(3):195–240. doi: 10.1017/s0033583597003351. [DOI] [PubMed] [Google Scholar]
  5. Arnez J. G., Harris D. C., Mitschler A., Rees B., Francklyn C. S., Moras D. Crystal structure of histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate. EMBO J. 1995 Sep 1;14(17):4143–4155. doi: 10.1002/j.1460-2075.1995.tb00088.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bajorath J., Stenkamp R., Aruffo A. Knowledge-based model building of proteins: concepts and examples. Protein Sci. 1993 Nov;2(11):1798–1810. doi: 10.1002/pro.5560021103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Banner D. W., Kokkinidis M., Tsernoglou D. Structure of the ColE1 rop protein at 1.7 A resolution. J Mol Biol. 1987 Aug 5;196(3):657–675. doi: 10.1016/0022-2836(87)90039-8. [DOI] [PubMed] [Google Scholar]
  8. Barrell B. G., Anderson S., Bankier A. T., de Bruijn M. H., Chen E., Coulson A. R., Drouin J., Eperon I. C., Nierlich D. P., Roe B. A. Different pattern of codon recognition by mammalian mitochondrial tRNAs. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3164–3166. doi: 10.1073/pnas.77.6.3164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Benson D. A., Boguski M., Lipman D. J., Ostell J. GenBank. Nucleic Acids Res. 1994 Sep;22(17):3441–3444. doi: 10.1093/nar/22.17.3441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  11. Biou V., Yaremchuk A., Tukalo M., Cusack S. The 2.9 A crystal structure of T. thermophilus seryl-tRNA synthetase complexed with tRNA(Ser). Science. 1994 Mar 11;263(5152):1404–1410. doi: 10.1126/science.8128220. [DOI] [PubMed] [Google Scholar]
  12. Buechter D. D., Schimmel P. Dissection of a class II tRNA synthetase: determinants for minihelix recognition are tightly associated with domain for amino acid activation. Biochemistry. 1993 May 18;32(19):5267–5272. doi: 10.1021/bi00070a039. [DOI] [PubMed] [Google Scholar]
  13. Buechter D. D., Schimmel P. Minor groove recognition of the critical acceptor helix base pair by an appended module of a class II tRNA synthetase. Biochemistry. 1995 May 9;34(18):6014–6019. doi: 10.1021/bi00018a002. [DOI] [PubMed] [Google Scholar]
  14. Burd C. G., Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. doi: 10.1126/science.8036511. [DOI] [PubMed] [Google Scholar]
  15. Calendar R., Berg P. The catalytic properties of tyrosyl ribonucleic acid synthetases from Escherichia coli and Bacillus subtilis. Biochemistry. 1966 May;5(5):1690–1695. doi: 10.1021/bi00869a034. [DOI] [PubMed] [Google Scholar]
  16. Conway N. E., McLaughlin L. W. The covalent attachment of multiple fluorophores to DNA containing phosphorothioate diesters results in highly sensitive detection of single-stranded DNA. Bioconjug Chem. 1991 Nov-Dec;2(6):452–457. doi: 10.1021/bc00012a013. [DOI] [PubMed] [Google Scholar]
  17. Cusack S., Berthet-Colominas C., Härtlein M., Nassar N., Leberman R. A second class of synthetase structure revealed by X-ray analysis of Escherichia coli seryl-tRNA synthetase at 2.5 A. Nature. 1990 Sep 20;347(6290):249–255. doi: 10.1038/347249a0. [DOI] [PubMed] [Google Scholar]
  18. Davis M. W., Buechter D. D., Schimmel P. Functional dissection of a predicted class-defining motif in a class II tRNA synthetase of unknown structure. Biochemistry. 1994 Aug 23;33(33):9904–9911. doi: 10.1021/bi00199a012. [DOI] [PubMed] [Google Scholar]
  19. Delarue M., Poterszman A., Nikonov S., Garber M., Moras D., Thierry J. C. Crystal structure of a prokaryotic aspartyl tRNA-synthetase. EMBO J. 1994 Jul 15;13(14):3219–3229. doi: 10.1002/j.1460-2075.1994.tb06623.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Eriani G., Delarue M., Poch O., Gangloff J., Moras D. Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature. 1990 Sep 13;347(6289):203–206. doi: 10.1038/347203a0. [DOI] [PubMed] [Google Scholar]
  21. Fersht A. R., Ashford J. S., Bruton C. J., Jakes R., Koch G. L., Hartley B. S. Active site titration and aminoacyl adenylate binding stoichiometry of aminoacyl-tRNA synthetases. Biochemistry. 1975 Jan 14;14(1):1–4. doi: 10.1021/bi00672a001. [DOI] [PubMed] [Google Scholar]
  22. Francklyn C., Schimmel P. Aminoacylation of RNA minihelices with alanine. Nature. 1989 Feb 2;337(6206):478–481. doi: 10.1038/337478a0. [DOI] [PubMed] [Google Scholar]
  23. Francklyn C., Shi J. P., Schimmel P. Overlapping nucleotide determinants for specific aminoacylation of RNA microhelices. Science. 1992 Feb 28;255(5048):1121–1125. doi: 10.1126/science.1546312. [DOI] [PubMed] [Google Scholar]
  24. Hill K., Schimmel P. Evidence that the 3' end of a tRNA binds to a site in the adenylate synthesis domain of an aminoacyl-tRNA synthetase. Biochemistry. 1989 Mar 21;28(6):2577–2586. doi: 10.1021/bi00432a035. [DOI] [PubMed] [Google Scholar]
  25. Ho C., Jasin M., Schimmel P. Amino acid replacements that compensate for a large polypeptide deletion in an enzyme. Science. 1985 Jul 26;229(4711):389–393. doi: 10.1126/science.3892692. [DOI] [PubMed] [Google Scholar]
  26. Hou Y. M., Schimmel P. A simple structural feature is a major determinant of the identity of a transfer RNA. Nature. 1988 May 12;333(6169):140–145. doi: 10.1038/333140a0. [DOI] [PubMed] [Google Scholar]
  27. Hou Y. M., Schimmel P. Evidence that a major determinant for the identity of a transfer RNA is conserved in evolution. Biochemistry. 1989 Aug 22;28(17):6800–6804. doi: 10.1021/bi00443a003. [DOI] [PubMed] [Google Scholar]
  28. Hyde C. C., Ahmed S. A., Padlan E. A., Miles E. W., Davies D. R. Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium. J Biol Chem. 1988 Nov 25;263(33):17857–17871. [PubMed] [Google Scholar]
  29. Jasin M., Regan L., Schimmel P. Modular arrangement of functional domains along the sequence of an aminoacyl tRNA synthetase. Nature. 1983 Dec 1;306(5942):441–447. doi: 10.1038/306441a0. [DOI] [PubMed] [Google Scholar]
  30. Jasin M., Regan L., Schimmel P. Two mutations in the dispensable part of alanine tRNA synthetase which affect the catalytic activity. J Biol Chem. 1985 Feb 25;260(4):2226–2230. [PubMed] [Google Scholar]
  31. Jasin M., Schimmel P. Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments. J Bacteriol. 1984 Aug;159(2):783–786. doi: 10.1128/jb.159.2.783-786.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Logan D. T., Mazauric M. H., Kern D., Moras D. Crystal structure of glycyl-tRNA synthetase from Thermus thermophilus. EMBO J. 1995 Sep 1;14(17):4156–4167. doi: 10.1002/j.1460-2075.1995.tb00089.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lu Y., Hill K. A. The invariant arginine in motif 2 of Escherichia coli alanyl-tRNA synthetase is important for catalysis but not for substrate binding. J Biol Chem. 1994 Apr 22;269(16):12137–12141. [PubMed] [Google Scholar]
  35. McClain W. H., Foss K. Changing the identity of a tRNA by introducing a G-U wobble pair near the 3' acceptor end. Science. 1988 May 6;240(4853):793–796. doi: 10.1126/science.2452483. [DOI] [PubMed] [Google Scholar]
  36. Moras D. Structural and functional relationships between aminoacyl-tRNA synthetases. Trends Biochem Sci. 1992 Apr;17(4):159–164. doi: 10.1016/0968-0004(92)90326-5. [DOI] [PubMed] [Google Scholar]
  37. Mosyak L., Reshetnikova L., Goldgur Y., Delarue M., Safro M. G. Structure of phenylalanyl-tRNA synthetase from Thermus thermophilus. Nat Struct Biol. 1995 Jul;2(7):537–547. doi: 10.1038/nsb0795-537. [DOI] [PubMed] [Google Scholar]
  38. Musier-Forsyth K., Schimmel P. Acceptor helix interactions in a class II tRNA synthetase: photoaffinity cross-linking of an RNA miniduplex substrate. Biochemistry. 1994 Jan 25;33(3):773–779. doi: 10.1021/bi00169a019. [DOI] [PubMed] [Google Scholar]
  39. Nureki O., Vassylyev D. G., Katayanagi K., Shimizu T., Sekine S., Kigawa T., Miyazawa T., Yokoyama S., Morikawa K. Architectures of class-defining and specific domains of glutamyl-tRNA synthetase. Science. 1995 Mar 31;267(5206):1958–1965. doi: 10.1126/science.7701318. [DOI] [PubMed] [Google Scholar]
  40. Park S. J., Hou Y. M., Schimmel P. A single base pair affects binding and catalytic parameters in the molecular recognition of a transfer RNA. Biochemistry. 1989 Mar 21;28(6):2740–2746. doi: 10.1021/bi00432a056. [DOI] [PubMed] [Google Scholar]
  41. Puglisi J. D., Tinoco I., Jr Absorbance melting curves of RNA. Methods Enzymol. 1989;180:304–325. doi: 10.1016/0076-6879(89)80108-9. [DOI] [PubMed] [Google Scholar]
  42. Regan L., Bowie J., Schimmel P. Polypeptide sequences essential for RNA recognition by an enzyme. Science. 1987 Mar 27;235(4796):1651–1653. doi: 10.1126/science.2435005. [DOI] [PubMed] [Google Scholar]
  43. Ribas de Pouplana L., Buechter D. D., Davis M. W., Schimmel P. Idiographic representation of conserved domain of a class II tRNA synthetase of unknown structure. Protein Sci. 1993 Dec;2(12):2259–2262. doi: 10.1002/pro.5560021225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ribas de Pouplana L., Schimmel P. Reconstruction of quaternary structures of class II tRNA synthetases by rational mutagenensis of a conserved domain. Biochemistry. 1997 Dec 9;36(49):15041–15048. doi: 10.1021/bi971788+. [DOI] [PubMed] [Google Scholar]
  45. Ripmaster T. L., Shiba K., Schimmel P. Wide cross-species aminoacyl-tRNA synthetase replacement in vivo: yeast cytoplasmic alanine enzyme replaced by human polymyositis serum antigen. Proc Natl Acad Sci U S A. 1995 May 23;92(11):4932–4936. doi: 10.1073/pnas.92.11.4932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rost B., Sander C. Combining evolutionary information and neural networks to predict protein secondary structure. Proteins. 1994 May;19(1):55–72. doi: 10.1002/prot.340190108. [DOI] [PubMed] [Google Scholar]
  47. Rost B., Schneider R., Sander C. Protein fold recognition by prediction-based threading. J Mol Biol. 1997 Jul 18;270(3):471–480. doi: 10.1006/jmbi.1997.1101. [DOI] [PubMed] [Google Scholar]
  48. Ruff M., Krishnaswamy S., Boeglin M., Poterszman A., Mitschler A., Podjarny A., Rees B., Thierry J. C., Moras D. Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp). Science. 1991 Jun 21;252(5013):1682–1689. doi: 10.1126/science.2047877. [DOI] [PubMed] [Google Scholar]
  49. Schimmel P. R., Söll D. Aminoacyl-tRNA synthetases: general features and recognition of transfer RNAs. Annu Rev Biochem. 1979;48:601–648. doi: 10.1146/annurev.bi.48.070179.003125. [DOI] [PubMed] [Google Scholar]
  50. Schimmel P. Alanine transfer RNA synthetase: structure-function relationships and molecular recognition of transfer RNA. Adv Enzymol Relat Areas Mol Biol. 1990;63:233–270. doi: 10.1002/9780470123096.ch4. [DOI] [PubMed] [Google Scholar]
  51. Shi J. P., Musier-Forsyth K., Schimmel P. Region of a conserved sequence motif in a class II tRNA synthetase needed for transfer of an activated amino acid to an RNA substrate. Biochemistry. 1994 May 3;33(17):5312–5318. doi: 10.1021/bi00183a039. [DOI] [PubMed] [Google Scholar]
  52. Shiba K., Ripmaster T., Suzuki N., Nichols R., Plotz P., Noda T., Schimmel P. Human alanyl-tRNA synthetase: conservation in evolution of catalytic core and microhelix recognition. Biochemistry. 1995 Aug 22;34(33):10340–10349. doi: 10.1021/bi00033a004. [DOI] [PubMed] [Google Scholar]
  53. Stultz C. M., White J. V., Smith T. F. Structural analysis based on state-space modeling. Protein Sci. 1993 Mar;2(3):305–314. doi: 10.1002/pro.5560020302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yang S. W., Nash H. A. Specific photocrosslinking of DNA-protein complexes: identification of contacts between integration host factor and its target DNA. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12183–12187. doi: 10.1073/pnas.91.25.12183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yarus M., Berg P. Recognition of tRNA by aminoacyl tRNA synthetases. J Mol Biol. 1967 Sep 28;28(3):479–490. doi: 10.1016/s0022-2836(67)80098-6. [DOI] [PubMed] [Google Scholar]

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