Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1988 Feb 11;16(3):815–832. doi: 10.1093/nar/16.3.815

RNA-protein cross-linking in Escherichia coli 50S ribosomal subunits; determination of sites on 23S RNA that are cross-linked to proteins L2, L4, L24 and L27 by treatment with 2-iminothiolane.

H Gulle 1, E Hoppe 1, M Osswald 1, B Greuer 1, R Brimacombe 1, G Stöffler 1
PMCID: PMC334721  PMID: 3278299

Abstract

RNA-protein cross-links were introduced into E. coli 50S ribosomal subunits by treatment with 2-iminothiolane followed by mild ultraviolet irradiation. After partial digestion of the RNA, the cross-linked RNA-protein complexes were separated by our recently published three-step procedure. In cases where this separation was inadequate, a further purification step was introduced, involving affinity chromatography with antibodies to the ribosomal 50S proteins. Analysis of the isolated complexes enabled four new cross-link sites on the 23S RNA to be identified, as well as re-confirming several previously established sites. The new sites are as follows: Protein L2 is cross-linked within an oligonucleotide at positions 1818-1823 in the 23S RNA, protein L4 within positions 320-325, protein L24 within positions 99-107, and protein L27 within positions 2320-2323.

Full text

PDF
815

Images in this article

Selected References

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

  1. Brimacombe R., Stiege W. Structure and function of ribosomal RNA. Biochem J. 1985 Jul 1;229(1):1–17. doi: 10.1042/bj2290001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brosius J., Dull T. J., Noller H. F. Complete nucleotide sequence of a 23S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jan;77(1):201–204. doi: 10.1073/pnas.77.1.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Fisher D. E., Conner G. E., Reeves W. H., Blobel G., Kunkel H. G. Synthesis and assembly of human small nuclear ribonucleoproteins generated by cell-free translation. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6356–6360. doi: 10.1073/pnas.80.20.6356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Greuer B., Osswald M., Brimacombe R., Stöffler G. RNA-protein cross-linking in Escherichia coli 30S ribosomal subunits; determination of sites on 16S RNA that are cross-linked to proteins S3, S4, S7, S9, S10, S11, S17, S18 and S21 by treatment with bis-(2-chloroethyl)-methylamine. Nucleic Acids Res. 1987 Apr 24;15(8):3241–3255. doi: 10.1093/nar/15.8.3241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gulle H., Brimacombe R., Stöffler-Meilicke M., Stöffler G. A rapid immunological spot test for the identification of proteins in covalently linked protein-nucleic acid complexes. J Immunol Methods. 1987 Sep 24;102(2):183–186. doi: 10.1016/0022-1759(87)90075-5. [DOI] [PubMed] [Google Scholar]
  6. Kronvall G., Seal U. S., Finstad J., Williams R. C., Jr Phylogenetic insight into evolution of mammalian Fc fragment of gamma G globulin using staphylococcal protein A. J Immunol. 1970 Jan;104(1):140–147. [PubMed] [Google Scholar]
  7. Kyriatsoulis A., Maly P., Greuer B., Brimacombe R., Stöffler G., Frank R., Blöcker H. RNA-protein cross-linking in Escherichia coli ribosomal subunits: localization of sites on 16S RNA which are cross-linked to proteins S17 and S21 by treatment with 2-iminothiolane. Nucleic Acids Res. 1986 Feb 11;14(3):1171–1186. doi: 10.1093/nar/14.3.1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Smith K. C., Aplin R. T. A mixed photoproduct of uracil and cysteine (5-S-cysteine-6-hydrouracil). A possible model for the in vivo cross-linking of deoxyribonucleic acid and protein by ultraviolet light. Biochemistry. 1966 Jun;5(6):2125–2130. doi: 10.1021/bi00870a046. [DOI] [PubMed] [Google Scholar]
  10. Stiege W., Zwieb C., Brimacombe R. Precise localisation of three intra-RNA cross-links in 23S RNA and one in 5S RNA, induced by treatment of Escherichia coli 50S ribosomal subunits with bis-(2-chloroethyl)-methylamine. Nucleic Acids Res. 1982 Nov 25;10(22):7211–7229. doi: 10.1093/nar/10.22.7211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Thomas C. A., Jr, Saigo K., McLeod E., Ito J. The separation of DNA segments attached to proteins. Anal Biochem. 1979 Feb;93(1):158–166. [PubMed] [Google Scholar]
  12. Vasilenko S. K., Ryte V. C. [Isolation of highly purified ribonuclease from cobra (Naja oxiana) venom]. Biokhimiia. 1975 May-Jun;40(3):578–583. [PubMed] [Google Scholar]
  13. Volckaert G., Fiers W. Micro thin-layer techniques for rapid sequence analysis of 32P-labeled RNA: double digestion and pancreatic ribonuclease analyses. Anal Biochem. 1977 Nov;83(1):228–239. doi: 10.1016/0003-2697(77)90531-0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]

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

RESOURCES