Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1982 Dec 11;10(23):7657–7676. doi: 10.1093/nar/10.23.7657

Protein-RNA crosslinking in Escherichia coli 30S ribosomal subunits. Identification of a 16S rRNA fragment crosslinked to protein S12 by the use of the chemical crosslinking reagent 1-ethyl-3-dimethyl-aminopropylcarbodiimide.

C Chiaruttini, A Expert-Bezançon, D Hayes, B Ehresmann
PMCID: PMC327037  PMID: 6760129

Abstract

1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC) was used to cross-link 30S ribosomal proteins to 16S rRNA within the E. coli 3OS ribosomal subunit. Covalently linked complexes containing 30S proteins and 16S rRNA, isolated by sedimentation of dissociated crosslinked 30S subunits through SDS containing sucrose gradients, were digested with RNase T1, and the resulting oligonucleotide-protein complexes were fractionated on SDS containing polyacrylamide gels. Eluted complexes containing 30S proteins S9 and S12 linked to oligonucleotides were obtained in pure form. Oligonucleotide 5'terminal labelling was successful in the case of S12 containing but not of the S9 containing complex and led to identification of the S12 bound oligonucleotide as CAACUCG which is located at positions 1316-1322 in the 16S rRNA sequence. Protein S12 is crosslinked to the terminal G of this heptanucleotide.

Full text

PDF
7657

Images in this article

7664Image
on p.7664
7666Image
on p.7666
7667Image
on p.7667
7668Image
on p.7668
7669Image
on p.7669
7670Image
on p.7670
7671Image
on p.7671
7672Image
on p.7672

Selected References

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

  1. Boguski M. S., Hieter P. A., Levy C. C. Identification of a cytidine-specific ribonuclease from chicken liver. J Biol Chem. 1980 Mar 10;255(5):2160–2163. [PubMed] [Google Scholar]
  2. Bäumert H. G., Sköld S. E., Kurland C. G. RNA-protein neighbourhoods of the ribosome obtained by crosslinking. Eur J Biochem. 1978 Sep 1;89(2):353–359. doi: 10.1111/j.1432-1033.1978.tb12536.x. [DOI] [PubMed] [Google Scholar]
  3. Carbon P., Ehresmann C., Ehresmann B., Ebel J. P. The sequence of Escherichia coli ribosomal 16 S RNA determined by new rapid gel methods. FEBS Lett. 1978 Oct 1;94(1):152–156. doi: 10.1016/0014-5793(78)80926-0. [DOI] [PubMed] [Google Scholar]
  4. Chiarrutini C., Expert-Bezançon A. RNA-protein crosslinking by reaction with a soluble carbodiimide. FEBS Lett. 1980 Sep 22;119(1):145–149. doi: 10.1016/0014-5793(80)81017-9. [DOI] [PubMed] [Google Scholar]
  5. Combarnous Y., Hennen G. Luteinizing hormone derivatives with covalently-linked subunits. FEBS Lett. 1974 Aug 25;44(2):224–228. doi: 10.1016/0014-5793(74)80730-1. [DOI] [PubMed] [Google Scholar]
  6. Cooperman B. S., Expert-Bezançon A., Kahan L., Dondon J., Grunberg-Manago M. IF-3 crosslinking to Escherichia coli ribosomal 30 S subunits by three different light-dependent procedures: identification of 30 S proteins crosslinked to IF-3--utilization of a new two-stage crosslinking reagent, p-nitrobenzylmaleimide. Arch Biochem Biophys. 1981 May;208(2):554–562. doi: 10.1016/0003-9861(81)90544-0. [DOI] [PubMed] [Google Scholar]
  7. Czernilofsky A. P., Kurland C. G., Stöffler G. 30S ribosomal proteins associated with the 3'-terminus of 16S RNA. FEBS Lett. 1975 Oct 15;58(1):281–284. doi: 10.1016/0014-5793(75)80279-1. [DOI] [PubMed] [Google Scholar]
  8. Dabbs E. R., Wittman H. G. A strain of Escherichia coli which gives rise to mutations in a large number of ribosomal proteins. Mol Gen Genet. 1976 Dec 22;149(3):303–309. doi: 10.1007/BF00268532. [DOI] [PubMed] [Google Scholar]
  9. Donis-Keller H., Maxam A. M., Gilbert W. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977 Aug;4(8):2527–2538. doi: 10.1093/nar/4.8.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Donis-Keller H. Phy M: an RNase activity specific for U and A residues useful in RNA sequence analysis. Nucleic Acids Res. 1980 Jul 25;8(14):3133–3142. doi: 10.1093/nar/8.14.3133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ehresmann B., Backendorf C., Ehresmann C., Ebel J. P. Characterization of the regions from E. coli 16 S RNA covalently linked to ribosomal proteins S4 and S20 after ultraviolet irradiation. FEBS Lett. 1977 Jun 15;78(2):261–266. doi: 10.1016/0014-5793(77)80319-0. [DOI] [PubMed] [Google Scholar]
  12. Expert-Bezançon A., Barritault D., Milet M., Guérin M. F., Hayes D. H. Identification of neighbouring proteins in Escherichia coli 30 S ribosome subunits. J Mol Biol. 1977 Jun 5;112(4):603–629. doi: 10.1016/s0022-2836(77)80166-6. [DOI] [PubMed] [Google Scholar]
  13. Expert-Bezançon A., Hayes D. Synthesis and properties of N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine, a new reagent for RNA-RNA and RNA-protein cross-linking. Eur J Biochem. 1980 Jan;103(2):365–375. doi: 10.1111/j.1432-1033.1980.tb04323.x. [DOI] [PubMed] [Google Scholar]
  14. GREENWOOD F. C., HUNTER W. M., GLOVER J. S. THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. Biochem J. 1963 Oct;89:114–123. doi: 10.1042/bj0890114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Glotz C., Brimacombe R. An experimentally-derived model for the secondary structure of the 16S ribosomal RNA from Escherichia coli. Nucleic Acids Res. 1980 Jun 11;8(11):2377–2395. doi: 10.1093/nar/8.11.2377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Golinska B., Millon R., Backendorf C., Olomucki M., Ebel J. P., Ehresmann B. Identification of a 16-S RNA fragment crosslinked to protein S1 within Escherichia coli ribosomal 30-S subunits by the use of a crosslinking reagent: ethyl 4-azidobenzoylaminoacetimidate. Eur J Biochem. 1981 Apr;115(3):479–484. doi: 10.1111/j.1432-1033.1981.tb06227.x. [DOI] [PubMed] [Google Scholar]
  17. Hardy S. J., Kurland C. G., Voynow P., Mora G. The ribosomal proteins of Escherichia coli. I. Purification of the 30S ribosomal proteins. Biochemistry. 1969 Jul;8(7):2897–2905. doi: 10.1021/bi00835a031. [DOI] [PubMed] [Google Scholar]
  18. Kurzer F., Douraghi-Zadeh K. Advances in the chemistry of carbodiimides. Chem Rev. 1967 Apr;67(2):107–152. doi: 10.1021/cr60246a001. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Lambert J. M., Traut R. R. The subunit interface of the Escherichia coli ribosome. Identification of proteins at the interface between the 30 S and 50 S subunits by crosslinking with 2-iminothiolane. J Mol Biol. 1981 Jul 5;149(3):451–476. doi: 10.1016/0022-2836(81)90481-2. [DOI] [PubMed] [Google Scholar]
  21. Laskey R. A., Mills A. D. Enhanced autoradiographic detection of 32P and 125I using intensifying screens and hypersensitized film. FEBS Lett. 1977 Oct 15;82(2):314–316. doi: 10.1016/0014-5793(77)80609-1. [DOI] [PubMed] [Google Scholar]
  22. Lockard R. E., Alzner-Deweerd B., Heckman J. E., MacGee J., Tabor M. W., RajBhandary U. L. Sequence analysis of 5'[32P] labeled mRNA and tRNA using polyacrylamide gel electrophoresis. Nucleic Acids Res. 1978 Jan;5(1):37–56. doi: 10.1093/nar/5.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lutter L. C., Ortanderl F., Fasold H. The use of a new series of cleavable protein-crosslinkers on the Escherichia coli ribosome. FEBS Lett. 1974 Nov 15;48(2):288–292. doi: 10.1016/0014-5793(74)80488-6. [DOI] [PubMed] [Google Scholar]
  24. MacKeen L. A., Kahan L., Wahba A. J., Schwartz I. Photochemical cross-linking of initiation factor-3 to Escherichia coli 30 S ribosomal subunits. J Biol Chem. 1980 Nov 10;255(21):10526–10531. [PubMed] [Google Scholar]
  25. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Millon R., Ebel J. P., Le Goffic F., Ehresmann B. Ribonucleic acid-protein crosslinking in Escherichia coli ribosomal 30S subunits by the use of two new heterobifunctional reagents: 4-azido-2,3,5,6-tetrafluoropyridine and 4-azido-3,5-dichloro-2,6-difluoropyridine. Biochem Biophys Res Commun. 1981 Aug 14;101(3):784–791. doi: 10.1016/0006-291x(81)91819-2. [DOI] [PubMed] [Google Scholar]
  27. Millon R., Olomucki M., Le Gall J. Y., Golinska B., Ebel J. P., Ehresmann B. Synthesis of a new reagent, ethyl 4-azidobenzoylaminoacetimidate, and its use for RNA-protein cross-linking within Escherichia coli ribosomal 30-S subunits. Eur J Biochem. 1980 Sep;110(2):485–492. doi: 10.1111/j.1432-1033.1980.tb04890.x. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Möller K., Rinke J., Ross A., Buddle G., Brimacombe R. The use of formaldehyde in RNA-protein cross-linking studies with ribosomal subunits from Escherichia coli. Eur J Biochem. 1977 Jun 1;76(1):175–187. doi: 10.1111/j.1432-1033.1977.tb11583.x. [DOI] [PubMed] [Google Scholar]
  30. Müller R., Garrett R. A., Noller H. F. The structure of the RNA binding site of ribosomal proteins S8 and S15. J Biol Chem. 1979 May 25;254(10):3873–3878. [PubMed] [Google Scholar]
  31. Oste C., Brimacombe R. The use of sym-triazine trichloride in RNA-protein cross-linking studies with Escherichia coli ribosomal subunits. Mol Gen Genet. 1979 Jan 5;168(1):81–86. doi: 10.1007/BF00267936. [DOI] [PubMed] [Google Scholar]
  32. Ramakrishnan V. R., Yabuki S., Sillers I. Y., Schindler D. G., Engelman D. M., Moore P. B. Positions of proteins S6, S11 and S15 in the 30 S ribosomal subunit of Escherichia coli. J Mol Biol. 1981 Dec 15;153(3):739–760. doi: 10.1016/0022-2836(81)90416-2. [DOI] [PubMed] [Google Scholar]
  33. Rinke J., Meinke M., Brimacombe R., Fink G., Rommel W., Fasold H. The use of azidoarylimidoesters in RNA-protein cross-linking studies with Escherichia coli ribosomes. J Mol Biol. 1980 Mar 5;137(3):301–304. doi: 10.1016/0022-2836(80)90318-6. [DOI] [PubMed] [Google Scholar]
  34. Silberklang M., Gillum A. M., RajBhandary U. L. The use of nuclease P1 in sequence analysis of end group labeled RNA. Nucleic Acids Res. 1977 Dec;4(12):4091–4108. doi: 10.1093/nar/4.12.4091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stiegler P., Carbon P., Ebel J. P., Ehresmann C. A general secondary-structure model for procaryotic and eucaryotic RNAs from the small ribosomal subunits. Eur J Biochem. 1981 Dec;120(3):487–495. doi: 10.1111/j.1432-1033.1981.tb05727.x. [DOI] [PubMed] [Google Scholar]
  36. Stiegler P., Carbon P., Zuker M., Ebel J. P., Ehresmann C. Structural organization of the 16S ribosomal RNA from E. coli. Topography and secondary structure. Nucleic Acids Res. 1981 May 11;9(9):2153–2172. doi: 10.1093/nar/9.9.2153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ulmer E., Meinke M., Ross A., Fink G., Brimacombe R. Chemical cross-linking of protein to RNA within intact ribosomal subunits from Escherichia coli. Mol Gen Genet. 1978 Apr 6;160(2):183–193. doi: 10.1007/BF00267480. [DOI] [PubMed] [Google Scholar]
  38. Woese C. R., Magrum L. J., Gupta R., Siegel R. B., Stahl D. A., Kop J., Crawford N., Brosius J., Gutell R., Hogan J. J. Secondary structure model for bacterial 16S ribosomal RNA: phylogenetic, enzymatic and chemical evidence. Nucleic Acids Res. 1980 May 24;8(10):2275–2293. doi: 10.1093/nar/8.10.2275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zimmermann R. A., Singh-Bergmann K. Binding sites for ribosomal proteins S8 and S15 in the 16 S RNA of Escherichia coli. Biochim Biophys Acta. 1979 Jul 26;563(2):422–431. doi: 10.1016/0005-2787(79)90061-3. [DOI] [PubMed] [Google Scholar]
  40. Zwieb C., Brimacombe R. Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin-Dahlem, GFR. Nucleic Acids Res. 1979;6(5):1775–1790. doi: 10.1093/nar/6.5.1775. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES