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. 1988 Mar 25;16(6):2369–2388. doi: 10.1093/nar/16.6.2369

Covalent cross-linking of poly(A) to Escherichia coli ribosomes, and localization of the cross-link site within the 16S RNA.

W Stiege 1, K Stade 1, D Schüler 1, R Brimacombe 1
PMCID: PMC336378  PMID: 3283702

Abstract

Poly(A) can be cross-linked to E. coli 70S ribosomes in the presence of tRNALys by mild ultraviolet irradiation. The cross-linking reaction is exclusively with the 30S subunit, and involves primarily the RNA moiety. Following a partial nuclease digestion, cross-linked complexes containing poly(A) and fragments of the 16S RNA were isolated by affinity chromatography on oligo(dT)-cellulose. The complexes were purified by gel electrophoresis and subjected to oligonucleotide analysis, which revealed a single cross-link site within positions 1394-1399 of the 16S RNA. The same pattern of cross-linking, at about one-fifth of the intensity, was observed in the absence of tRNALys. The cross-link site to poly(A), together with other sites in the 16S RNA that have been implicated in ribosomal function, is discussed in the framework of our recent model for the three-dimensional structure of 16S RNA; all of the functional sites are clustered together in two distinct groups in the model.

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Selected References

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

  1. Atmadja J., Brimacombe R., Blöcker H., Frank R. Investigation of the tertiary folding of Escherichia coli 16S RNA by in situ intra-RNA cross-linking within 30S ribosomal subunits. Nucleic Acids Res. 1985 Oct 11;13(19):6919–6936. doi: 10.1093/nar/13.19.6919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Babkina G. T., Veniaminova A. G., Vladimirov S. N., Karpova G. G., Yamkovoy V. I., Berzin V. A., Gren E. J., Cielens I. E. Affinity labelling of Escherichia coli ribosomes with a benzylidene derivative of AUGU6 within initiation and pretranslocational complexes. FEBS Lett. 1986 Jul 7;202(2):340–344. doi: 10.1016/0014-5793(86)80714-1. [DOI] [PubMed] [Google Scholar]
  3. Brimacombe R., Atmadja J., Stiege W., Schüler D. A detailed model of the three-dimensional structure of Escherichia coli 16 S ribosomal RNA in situ in the 30 S subunit. J Mol Biol. 1988 Jan 5;199(1):115–136. doi: 10.1016/0022-2836(88)90383-x. [DOI] [PubMed] [Google Scholar]
  4. Brimacombe R., Maly P., Zwieb C. The structure of ribosomal RNA and its organization relative to ribosomal protein. Prog Nucleic Acid Res Mol Biol. 1983;28:1–48. doi: 10.1016/s0079-6603(08)60081-1. [DOI] [PubMed] [Google Scholar]
  5. Brosius J., Palmer M. L., Kennedy P. J., Noller H. F. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4801–4805. doi: 10.1073/pnas.75.10.4801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carbon P., Ehresmann C., Ehresmann B., Ebel J. P. The complete nucleotide sequence of the ribosomal 16-S RNA from Excherichia coli. Experimental details and cistron heterogeneities. Eur J Biochem. 1979 Oct 15;100(2):399–410. doi: 10.1111/j.1432-1033.1979.tb04183.x. [DOI] [PubMed] [Google Scholar]
  7. Ciesiolka J., Gornicki P., Ofengand J. Identification of the site of cross-linking in 16S rRNA of an aromatic azide photoaffinity probe attached to the 5'-anticodon base of A site bound tRNA. Biochemistry. 1985 Aug 27;24(18):4931–4938. doi: 10.1021/bi00339a031. [DOI] [PubMed] [Google Scholar]
  8. Ehresmann C., Ofengand J. Two-dimensional gel electrophoresis technique for determination of the cross-linked nucleotides in cleavable covalent RNA-RNA complexes. Application to Escherichia coli and Bacillus subtilis acetylvalyl-tRNA covalently linked to E. coli 16S and yeast 18S ribosomal RNA. Biochemistry. 1984 Jan 31;23(3):438–445. doi: 10.1021/bi00298a007. [DOI] [PubMed] [Google Scholar]
  9. Evstafieva A. G., Shatsky I. N., Bogdanov A. A., Semenkov Y. P., Vasiliev V. D. Localization of 5' and 3' ends of the ribosome-bound segment of template polynucleotides by immune electron microscopy. EMBO J. 1983;2(5):799–804. doi: 10.1002/j.1460-2075.1983.tb01503.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Girshovich A. S., Bochkareva E. S., Vasiliev V. D. Localization of elongation factor Tu on the ribosome. FEBS Lett. 1986 Mar 3;197(1-2):192–198. doi: 10.1016/0014-5793(86)80325-8. [DOI] [PubMed] [Google Scholar]
  11. Gornicki P., Nurse K., Hellmann W., Boublik M., Ofengand J. High resolution localization of the tRNA anticodon interaction site on the Escherichia coli 30 S ribosomal subunit. J Biol Chem. 1984 Aug 25;259(16):10493–10498. [PubMed] [Google Scholar]
  12. Kang C. W., Cantor C. R. Structure of ribosome-bound messenger RNA as revealed by enzymatic accessibility studies. J Mol Biol. 1985 Jan 20;181(2):241–251. doi: 10.1016/0022-2836(85)90088-9. [DOI] [PubMed] [Google Scholar]
  13. Lake J. A. Aminoacyl-tRNA binding at the recognition site is the first step of the elongation cycle of protein synthesis. Proc Natl Acad Sci U S A. 1977 May;74(5):1903–1907. doi: 10.1073/pnas.74.5.1903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Langer J. A., Lake J. A. Elongation factor Tu localized on the exterior surface of the small ribosomal subunit. J Mol Biol. 1986 Feb 20;187(4):617–621. doi: 10.1016/0022-2836(86)90339-6. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Moazed D., Noller H. F. Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature. 1987 Jun 4;327(6121):389–394. doi: 10.1038/327389a0. [DOI] [PubMed] [Google Scholar]
  17. Moazed D., Noller H. F. Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes. Cell. 1986 Dec 26;47(6):985–994. doi: 10.1016/0092-8674(86)90813-5. [DOI] [PubMed] [Google Scholar]
  18. Morgan J., Brimacombe R. A series of specific ribonucleoprotein fragments from the 30-S subparticle of Escherichia coli ribosomes. Eur J Biochem. 1972 Sep 25;29(3):542–552. doi: 10.1111/j.1432-1033.1972.tb02020.x. [DOI] [PubMed] [Google Scholar]
  19. Osswald M., Greuer B., Brimacombe R., Stöffler G., Bäumert H., Fasold H. RNA-protein cross-linking in Escherichia coli 30S ribosomal subunits; determination of sites on 16S RNA that are cross-linked to proteins S3, S4, S5, S7, S8, S9, S11, S13, S19 and S21 by treatment with methyl p-azidophenyl acetimidate. Nucleic Acids Res. 1987 Apr 24;15(8):3221–3240. doi: 10.1093/nar/15.8.3221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Prince J. B., Taylor B. H., Thurlow D. L., Ofengand J., Zimmermann R. A. Covalent crosslinking of tRNA1Val to 16S RNA at the ribosomal P site: identification of crosslinked residues. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5450–5454. doi: 10.1073/pnas.79.18.5450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rheinberger H. J., Nierhaus K. H. Allosteric interactions between the ribosomal transfer RNA-binding sites A and E. J Biol Chem. 1986 Jul 15;261(20):9133–9139. [PubMed] [Google Scholar]
  22. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Spirin A. S. Location of tRNA on the ribosome. FEBS Lett. 1983 Jun 13;156(2):217–221. doi: 10.1016/0014-5793(83)80499-2. [DOI] [PubMed] [Google Scholar]
  24. Stahl J., Karpova G. G. Investigations on the messenger RNA binding site of eukaryotic ribosomes by using reactive oligo(U) derivatives. Biomed Biochim Acta. 1985;44(7-8):1057–1064. [PubMed] [Google Scholar]
  25. Stiege W., Atmadja J., Zobawa M., Brimacombe R. Investigation of the tertiary folding of Escherichia coli ribosomal RNA by intra-RNA cross-linking in vivo. J Mol Biol. 1986 Sep 5;191(1):135–138. doi: 10.1016/0022-2836(86)90429-8. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Towbin H., Elson D. A photoaffinity labelling study of the messenger RNA-binding region of Escherichia coli ribosomes. Nucleic Acids Res. 1978 Sep;5(9):3389–3407. doi: 10.1093/nar/5.9.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Trempe M. R., Ohgi K., Glitz D. G. Ribosome structure. Localization of 7-methylguanosine in the small subunits of Escherichia coli and chloroplast ribosomes by immunoelectron microscopy. J Biol Chem. 1982 Aug 25;257(16):9822–9829. [PubMed] [Google Scholar]
  29. Vassilenko S. K., Carbon P., Ebel J. P., Ehresmann C. Topography of 16 S RNA in 30 S subunits and 70 S ribosomes accessibility to cobra venom ribonuclease. J Mol Biol. 1981 Nov 15;152(4):699–721. doi: 10.1016/0022-2836(81)90123-6. [DOI] [PubMed] [Google Scholar]
  30. Volckaert G., Fiers W. A micromethod for base analysis of 32P-labeled oligoribonulcleotides. Anal Biochem. 1977 Nov;83(1):222–227. doi: 10.1016/0003-2697(77)90530-9. [DOI] [PubMed] [Google Scholar]
  31. Wagner R., Gassen H. G. Identification of a 16S rna sequence located in the decoding site of 30S ribosomes. FEBS Lett. 1976 Sep 1;67(3):312–315. doi: 10.1016/0014-5793(76)80554-6. [DOI] [PubMed] [Google Scholar]
  32. Wagner R., Gassen H. G. On the covalent binding of mRNA models to the part of the 16 S RNA which is located in the mRNA binding site of the 30 S ribosome. Biochem Biophys Res Commun. 1975 Jul 22;65(2):519–529. doi: 10.1016/s0006-291x(75)80178-1. [DOI] [PubMed] [Google Scholar]
  33. Yuki A., Brimacombe R. Nucleotide sequences of Escherichia coli 16-S RNA associated with ribosomal proteins S7, S9, S10, S14 and S19. Eur J Biochem. 1975 Aug 1;56(1):23–34. doi: 10.1111/j.1432-1033.1975.tb02203.x. [DOI] [PubMed] [Google Scholar]

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