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. 1995 Sep 15;14(18):4578–4588. doi: 10.1002/j.1460-2075.1995.tb00137.x

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies.

H Urlaub 1, V Kruft 1, O Bischof 1, E C Müller 1, B Wittmann-Liebold 1
PMCID: PMC394550  PMID: 7556101

Abstract

We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bergmann U., Wittmann-Liebold B. Identification of cross-linked amino acids in the protein pair HmaL23-HmaL29 from the 50S ribosomal subunit of the archaebacterium Haloarcula marismortui. Biochemistry. 1993 Mar 23;32(11):2880–2887. doi: 10.1021/bi00062a020. [DOI] [PubMed] [Google Scholar]
  2. Bischof O., Kruft V., Wittmann-Liebold B. Analysis of the puromycin binding site in the 70 S ribosome of Escherichia coli at the peptide level. J Biol Chem. 1994 Jul 15;269(28):18315–18319. [PubMed] [Google Scholar]
  3. Brauer D., Ochsner I. The primary structure of protein L1 from the large ribosomal subunit of Escherichia coli. FEBS Lett. 1978 Dec 15;96(2):317–321. doi: 10.1016/0014-5793(78)80426-8. [DOI] [PubMed] [Google Scholar]
  4. Brauer D., Röming R. The primary structure of protein S3 from the small ribosomal subunit of Escherichia coli. FEBS Lett. 1979 Oct 15;106(2):352–357. doi: 10.1016/0014-5793(79)80531-1. [DOI] [PubMed] [Google Scholar]
  5. Brimacombe R. RNA-protein interactions in the Escherichia coli ribosome. Biochimie. 1991 Jul-Aug;73(7-8):927–936. doi: 10.1016/0300-9084(91)90134-m. [DOI] [PubMed] [Google Scholar]
  6. Brockmöller J., Kamp R. M. Cross-linked amino acids in the protein pair S13-S19 and sequence analysis of protein S13 of Bacillus stearothermophilus ribosomes. Biochemistry. 1988 May 3;27(9):3372–3381. doi: 10.1021/bi00409a038. [DOI] [PubMed] [Google Scholar]
  7. Brockmöller J., Kamp R. M. Isolation and identification of two protein-protein crosslinks within the two subunits of Bacillus stearothermophilus ribosomes. Biol Chem Hoppe Seyler. 1986 Sep;367(9):925–935. doi: 10.1515/bchm3.1986.367.2.925. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Chan Y. L., Suzuki K., Olvera J., Wool I. G. Zinc finger-like motifs in rat ribosomal proteins S27 and S29. Nucleic Acids Res. 1993 Feb 11;21(3):649–655. doi: 10.1093/nar/21.3.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen R., Mende L., Arfsten U. The primary structure of protein L27 from the peptidyl-tRNA binding site of Escherichia coli ribosomes. FEBS Lett. 1975 Nov 1;59(1):96–99. doi: 10.1016/0014-5793(75)80349-8. [DOI] [PubMed] [Google Scholar]
  11. Chen R., Wittmann-Liebold B. The primary structure of protein S9 from the 30S subunit of Escherichia coli ribosomes. FEBS Lett. 1975 Mar 15;52(1):139–140. doi: 10.1016/0014-5793(75)80656-9. [DOI] [PubMed] [Google Scholar]
  12. Chittum H. S., Champney W. S. Ribosomal protein gene sequence changes in erythromycin-resistant mutants of Escherichia coli. J Bacteriol. 1994 Oct;176(20):6192–6198. doi: 10.1128/jb.176.20.6192-6198.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Döring T., Mitchell P., Osswald M., Bochkariov D., Brimacombe R. The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop. EMBO J. 1994 Jun 1;13(11):2677–2685. doi: 10.1002/j.1460-2075.1994.tb06558.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ehresmann B., Reinbolt J., Backendorf C., Tritsch D., Ebel J. Studies of the binding sites of Escherichia coli ribosomal protein S7 with 16S RNA by ultraviolet irradiation. FEBS Lett. 1976 Sep 1;67(3):316–319. doi: 10.1016/0014-5793(76)80555-8. [DOI] [PubMed] [Google Scholar]
  15. Engebrecht J., Roeder G. S. MER1, a yeast gene required for chromosome pairing and genetic recombination, is induced in meiosis. Mol Cell Biol. 1990 May;10(5):2379–2389. doi: 10.1128/mcb.10.5.2379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Funatsu G., Yaguchi M., Wittmann-Liebold B. Primary stucture of protein S12 from the small Escherichia coli ribosomal subunit. FEBS Lett. 1977 Jan 15;73(1):12–17. doi: 10.1016/0014-5793(77)80004-5. [DOI] [PubMed] [Google Scholar]
  17. Gibson T. J., Thompson J. D., Heringa J. The KH domain occurs in a diverse set of RNA-binding proteins that include the antiterminator NusA and is probably involved in binding to nucleic acid. FEBS Lett. 1993 Jun 21;324(3):361–366. doi: 10.1016/0014-5793(93)80152-k. [DOI] [PubMed] [Google Scholar]
  18. Golden B. L., Hoffman D. W., Ramakrishnan V., White S. W. Ribosomal protein S17: characterization of the three-dimensional structure by 1H and 15N NMR. Biochemistry. 1993 Nov 30;32(47):12812–12820. doi: 10.1021/bi00210a033. [DOI] [PubMed] [Google Scholar]
  19. Golden B. L., Ramakrishnan V., White S. W. Ribosomal protein L6: structural evidence of gene duplication from a primitive RNA binding protein. EMBO J. 1993 Dec 15;12(13):4901–4908. doi: 10.1002/j.1460-2075.1993.tb06184.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Herfurth E., Briesemeister U., Wittmann-Liebold B. Complete amino acid sequence of ribosomal protein S14 from Bacillus stearothermophilus and homology studies to other ribosomal proteins. FEBS Lett. 1994 Aug 29;351(1):114–118. doi: 10.1016/0014-5793(94)00838-8. [DOI] [PubMed] [Google Scholar]
  21. Herfurth E., Hirano H., Wittmann-Liebold B. The amino-acid sequences of the Bacillus stearothermophilus ribosomal proteins S17 and S21 and their comparison to homologous proteins of other ribosomes. Biol Chem Hoppe Seyler. 1991 Oct;372(10):955–961. doi: 10.1515/bchm3.1991.372.2.955. [DOI] [PubMed] [Google Scholar]
  22. Herwig S., Kruft V., Eckart K., Wittmann-Liebold B. Cross-linked amino acids in the protein pairs L3-L19 and L23-L29 of Bacillus stearothermophilus ribosomes after treatment with diepoxybutane. J Biol Chem. 1993 Mar 5;268(7):4643–4650. [PubMed] [Google Scholar]
  23. Herwig S., Kruft V., Wittmann-Liebold B. Primary structures of ribosomal proteins L3 and L4 from Bacillus stearothermophilus. Eur J Biochem. 1992 Aug 1;207(3):877–885. doi: 10.1111/j.1432-1033.1992.tb17119.x. [DOI] [PubMed] [Google Scholar]
  24. Hoffman D. W., Davies C., Gerchman S. E., Kycia J. H., Porter S. J., White S. W., Ramakrishnan V. Crystal structure of prokaryotic ribosomal protein L9: a bi-lobed RNA-binding protein. EMBO J. 1994 Jan 1;13(1):205–212. doi: 10.1002/j.1460-2075.1994.tb06250.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hoffman D. W., Query C. C., Golden B. L., White S. W., Keene J. D. RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2495–2499. doi: 10.1073/pnas.88.6.2495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kimura M., Kimura J., Ashman K. The complete primary structure of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus. Eur J Biochem. 1985 Aug 1;150(3):491–497. doi: 10.1111/j.1432-1033.1985.tb09049.x. [DOI] [PubMed] [Google Scholar]
  27. Kimura M., Kimura J., Watanabe K. The primary structure of ribosomal protein L2 from Bacillus stearothermophilus. Eur J Biochem. 1985 Dec 2;153(2):289–297. doi: 10.1111/j.1432-1033.1985.tb09299.x. [DOI] [PubMed] [Google Scholar]
  28. Kimura M. The nucleotide sequences of Bacillus stearothermophilus ribosomal protein S12 and S7 genes: comparison with the str operon of Escherichia coli. Agric Biol Chem. 1991 Jan;55(1):207–213. doi: 10.1271/bbb1961.55.207. [DOI] [PubMed] [Google Scholar]
  29. Koonin E. V., Bork P., Sander C. A novel RNA-binding motif in omnipotent suppressors of translation termination, ribosomal proteins and a ribosome modification enzyme? Nucleic Acids Res. 1994 Jun 11;22(11):2166–2167. doi: 10.1093/nar/22.11.2166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  31. Leijonmarck M., Eriksson S., Liljas A. Crystal structure of a ribosomal component at 2.6 A resolution. Nature. 1980 Aug 21;286(5775):824–826. doi: 10.1038/286824a0. [DOI] [PubMed] [Google Scholar]
  32. Lindahl M., Svensson L. A., Liljas A., Sedelnikova S. E., Eliseikina I. A., Fomenkova N. P., Nevskaya N., Nikonov S. V., Garber M. B., Muranova T. A. Crystal structure of the ribosomal protein S6 from Thermus thermophilus. EMBO J. 1994 Mar 15;13(6):1249–1254. doi: 10.2210/pdb1ris/pdb. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Maly P., Rinke J., Ulmer E., Zwieb C., Brimacombe R. Precise localization of the site of cross-linking between protein L4 and 23S ribonucleic acid induced by mild ultraviolet irradiation of Escherichia coli 50S ribosomal subunits. Biochemistry. 1980 Sep 2;19(18):4179–4188. doi: 10.1021/bi00559a007. [DOI] [PubMed] [Google Scholar]
  34. Mattaj I. W. RNA recognition: a family matter? Cell. 1993 Jun 4;73(5):837–840. doi: 10.1016/0092-8674(93)90265-r. [DOI] [PubMed] [Google Scholar]
  35. Morinaga T., Funatsu G., Funatsu M. Primary structure of protein L14 isolated from Escherichia coli ribosomes. FEBS Lett. 1978 Jul 1;91(1):74–77. doi: 10.1016/0014-5793(78)80020-9. [DOI] [PubMed] [Google Scholar]
  36. Möller K., Brimacombe R. Specific cross-linking of proteins S7 and L4 to ribosomal RNA, by UV irradiation of Escherichia coli ribosomal subunits. Mol Gen Genet. 1975 Dec 9;141(4):343–355. doi: 10.1007/BF00331455. [DOI] [PubMed] [Google Scholar]
  37. Möller K., Zwieb C., Brimacombe R. Identification of the oligonucleotide and oligopeptide involved in an RNA--protein crosslink induced by ultraviolet irradiation of Escherichia coli 30 S ribosomal subunits. J Mol Biol. 1978 Dec 15;126(3):489–506. doi: 10.1016/0022-2836(78)90055-4. [DOI] [PubMed] [Google Scholar]
  38. Nagai K., Oubridge C., Jessen T. H., Li J., Evans P. R. Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A. Nature. 1990 Dec 6;348(6301):515–520. doi: 10.1038/348515a0. [DOI] [PubMed] [Google Scholar]
  39. Newberry V., Yaguchi M., Garrett R. A. A trypsin-resistant fragment from complexes of ribosomal protein S4 with 16-S RNA of Escherichia coli and from the uncomplexed protein. Eur J Biochem. 1977 Jun 1;76(1):51–61. doi: 10.1111/j.1432-1033.1977.tb11569.x. [DOI] [PubMed] [Google Scholar]
  40. Oubridge C., Ito N., Evans P. R., Teo C. H., Nagai K. Crystal structure at 1.92 A resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin. Nature. 1994 Dec 1;372(6505):432–438. doi: 10.1038/372432a0. [DOI] [PubMed] [Google Scholar]
  41. Pohl T., Wittmann-Liebold B. Identification of a cross-link in the Escherichia coli ribosomal protein pair S13-S19 at the amino acid level. J Biol Chem. 1988 Mar 25;263(9):4293–4301. [PubMed] [Google Scholar]
  42. Ramakrishnan V., White S. W. The structure of ribosomal protein S5 reveals sites of interaction with 16S rRNA. Nature. 1992 Aug 27;358(6389):768–771. doi: 10.1038/358768a0. [DOI] [PubMed] [Google Scholar]
  43. Reinbolt J., Tritsch D. The primary structure of ribosomal protein S7 from E. coli strains K and B. FEBS Lett. 1978 Jul 15;91(2):297–301. doi: 10.1016/0014-5793(78)81196-x. [DOI] [PubMed] [Google Scholar]
  44. Romero D. P., Arredondo J. A., Traut R. R. Identification of a region of Escherichia coli ribosomal protein L2 required for the assembly of L16 into the 50 S ribosomal subunit. J Biol Chem. 1990 Oct 25;265(30):18185–18191. [PubMed] [Google Scholar]
  45. 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]
  46. Rost B., Sander C., Schneider R. PHD--an automatic mail server for protein secondary structure prediction. Comput Appl Biosci. 1994 Feb;10(1):53–60. doi: 10.1093/bioinformatics/10.1.53. [DOI] [PubMed] [Google Scholar]
  47. Rould M. A., Perona J. J., Söll D., Steitz T. A. Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution. Science. 1989 Dec 1;246(4934):1135–1142. doi: 10.1126/science.2479982. [DOI] [PubMed] [Google Scholar]
  48. Schiltz E., Reinbolt J. Determination of the complete amino-acid sequence of protein S4 from Escherichia coli ribosomes. Eur J Biochem. 1975 Aug 15;56(2):467–481. doi: 10.1111/j.1432-1033.1975.tb02253.x. [DOI] [PubMed] [Google Scholar]
  49. Siomi H., Matunis M. J., Michael W. M., Dreyfuss G. The pre-mRNA binding K protein contains a novel evolutionarily conserved motif. Nucleic Acids Res. 1993 Mar 11;21(5):1193–1198. doi: 10.1093/nar/21.5.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tanaka I., Kimura M., Kimura J., Dijk J. The amino acid sequence of two small ribosomal proteins from Bacillus stearothermophilus. FEBS Lett. 1984 Jan 30;166(2):343–346. doi: 10.1016/0014-5793(84)80109-x. [DOI] [PubMed] [Google Scholar]
  51. Traut R. R., Bollen A., Sun T. T., Hershey J. W., Sundberg J., Pierce L. R. Methyl 4-mercaptobutyrimidate as a cleavable cross-linking reagent and its application to the Escherichia coli 30S ribosome. Biochemistry. 1973 Aug 14;12(17):3266–3273. doi: 10.1021/bi00741a019. [DOI] [PubMed] [Google Scholar]
  52. Vandekerckhove J., Rombauts W., Peeters B., Wittmann-Liebold B. Determination of the complete amino acid sequence of protein S21 from Escherichia coli ribosomes. Hoppe Seylers Z Physiol Chem. 1975 Dec;356(12):1955–1976. doi: 10.1515/bchm2.1975.356.2.1955. [DOI] [PubMed] [Google Scholar]
  53. Watanabe K., Kimura M. Location of the binding region for 23S ribosomal RNA on ribosomal protein L2 from Bacillus stearothermophilus. Eur J Biochem. 1985 Dec 2;153(2):299–304. doi: 10.1111/j.1432-1033.1985.tb09300.x. [DOI] [PubMed] [Google Scholar]
  54. Wilson K. S., Appelt K., Badger J., Tanaka I., White S. W. Crystal structure of a prokaryotic ribosomal protein. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7251–7255. doi: 10.1073/pnas.83.19.7251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wittmann-Liebold B., Bosserhoff A. Primary structure of protein S2 from the Escherichia coli ribosome. FEBS Lett. 1981 Jun 29;129(1):10–16. doi: 10.1016/0014-5793(81)80743-0. [DOI] [PubMed] [Google Scholar]
  56. Wittmann-Liebold B., Marzinzig E. Primary structure of protein L28 from the large subunit of Escherichia coli ribosomes. FEBS Lett. 1977 Sep 1;81(1):214–217. doi: 10.1016/0014-5793(77)80960-5. [DOI] [PubMed] [Google Scholar]
  57. Wower I., Brimacombe R. The localization of multiple sites on 16S RNA which are cross-linked to proteins S7 and S8 in Escherichia coli 30S ribosomal subunits by treatment with 2-iminothiolane. Nucleic Acids Res. 1983 Mar 11;11(5):1419–1437. doi: 10.1093/nar/11.5.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wower I., Wower J., Meinke M., Brimacombe R. The use of 2-iminothiolane as an RNA-protein cross-linking agent in Escherichia coli ribosomes, and the localisation on 23S RNA of sites cross-linked to proteins L4, L6, L21, L23, L27 and L29. Nucleic Acids Res. 1981 Sep 11;9(17):4285–4302. doi: 10.1093/nar/9.17.4285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yaguchi M. Primary structure of protein S18 from the small Escherichia coli ribosomal subunit. FEBS Lett. 1975 Nov 15;59(2):217–220. doi: 10.1016/0014-5793(75)80378-4. [DOI] [PubMed] [Google Scholar]
  60. Yaguchi M., Wittmann H. G. The primary structure of protein S17 from the small ribosomal subunit of Escherichia coli. FEBS Lett. 1978 Mar 1;87(1):37–40. doi: 10.1016/0014-5793(78)80127-6. [DOI] [PubMed] [Google Scholar]

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