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. 1991 Feb 25;19(4):801–808. doi: 10.1093/nar/19.4.801

Neighbourhood of the central fold of the tRNA molecule bound to the E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop.

J Podkowinski 1, P Gornicki 1
PMCID: PMC333714  PMID: 1708125

Abstract

The neighbourhood of the dihydrouridine loop of tRNA molecule bound to E. coli ribosome has been studied by affinity labeling, using modified tRNAs carrying photoreactive azidonitrophenyl probes attached to the 3-(3-amino-3-carboxypropyl)-uridine located at position 20:1 of Lupin methionine elongator tRNA. The maximum distance between the pyrimidine ring and the azido group estimated for the two probes employed in this study is 10-11 A and 18-19 A, respectively. Cross-linking of the uncharged, modified tRNAs has been studied with poly(A, U, G) as a message, under conditions directing uncharged tRNAs preferentially to the ribosomal P-site. Modified tRNAs bind covalently to both ribosomal subunits with high yields upon irradiation of the respective non-covalent complexes. Proteins S7, L33 and L1 have been consistently found cross-linked to tRNAs modified with both probes, and S5 and L5 to tRNA modified with the longer probe. Surprisingly, an S5-tRNA cross-linking product is reproducibly found in a protein fraction prepared from the purified 50S subunit. Cross-linking to rRNAs is significant only for the longer probe and is stimulated 2-4 fold in the presence of poly(A,U,G). The cross-linking sites are located between nucleotides 1302 and 1398 in 16S rRNA and between nucleotides 2281 and 2358 in 23S 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. Abdurashidova G. G., Tsvetkova E. A., Budowsky E. I. Nucleotide residues of tRNA, directly interacting with proteins within the complex of the 30 S subunit of E. coli ribosome with poly(U) and NAcPhe-tRNA(Phe). FEBS Lett. 1989 Jan 30;243(2):299–302. doi: 10.1016/0014-5793(89)80149-8. [DOI] [PubMed] [Google Scholar]
  2. Barta A., Steiner G., Brosius J., Noller H. F., Kuechler E. Identification of a site on 23S ribosomal RNA located at the peptidyl transferase center. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3607–3611. doi: 10.1073/pnas.81.12.3607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boileau G., Butler P., Hershey J. W., Traut R. R. Direct cross-links between initiation factors 1, 2, and 3 and ribosomal proteins promoted by 2-iminothiolane. Biochemistry. 1983 Jun 21;22(13):3162–3170. doi: 10.1021/bi00282a020. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Ehresmann C., Moine H., Mougel M., Dondon J., Grunberg-Manago M., Ebel J. P., Ehresmann B. Cross-linking of initiation factor IF3 to Escherichia coli 30S ribosomal subunit by trans-diamminedichloroplatinum(II): characterization of two cross-linking sites in 16S rRNA; a possible way of functioning for IF3. Nucleic Acids Res. 1986 Jun 25;14(12):4803–4821. doi: 10.1093/nar/14.12.4803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gornicki P., Ciesiolka J., Ofengand J. Cross-linking of the anticodon of P and A site bound tRNAs to the ribosome via aromatic azides of variable length: involvement of 16S rRNA at the A site. Biochemistry. 1985 Aug 27;24(18):4924–4930. doi: 10.1021/bi00339a030. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Gulle H., Hoppe E., Osswald M., Greuer B., Brimacombe R., Stöffler G. 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. Nucleic Acids Res. 1988 Feb 11;16(3):815–832. doi: 10.1093/nar/16.3.815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hingerty B., Brown R. S., Jack A. Further refinement of the structure of yeast tRNAPhe. J Mol Biol. 1978 Sep 25;124(3):523–534. doi: 10.1016/0022-2836(78)90185-7. [DOI] [PubMed] [Google Scholar]
  12. Lin F. L., Kahan L., Ofengand J. Crosslinking of phenylalanyl-tRNA to the ribosomal A site via a photoaffinity probe attached to the 4-thiouridine residue is exclusively to ribosomal protein S19. J Mol Biol. 1984 Jan 5;172(1):77–86. doi: 10.1016/0022-2836(84)90415-7. [DOI] [PubMed] [Google Scholar]
  13. Mitchell P., Osswald M., Schueler D., Brimacombe R. Selective isolation and detailed analysis of intra-RNA cross-links induced in the large ribosomal subunit of E. coli: a model for the tertiary structure of the tRNA binding domain in 23S RNA. Nucleic Acids Res. 1990 Aug 11;18(15):4325–4333. doi: 10.1093/nar/18.15.4325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Moazed D., Noller H. F. Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA. J Mol Biol. 1990 Jan 5;211(1):135–145. doi: 10.1016/0022-2836(90)90016-F. [DOI] [PubMed] [Google Scholar]
  15. Moazed D., Noller H. F. Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites. Cell. 1989 May 19;57(4):585–597. doi: 10.1016/0092-8674(89)90128-1. [DOI] [PubMed] [Google Scholar]
  16. Podkowiński J., Górnicki P. Ribosomal proteins S7 and L1 are located close to the decoding site of E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached adjacent to the 3'-end of the anticodon. Nucleic Acids Res. 1989 Nov 11;17(21):8767–8782. doi: 10.1093/nar/17.21.8767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Pon C. L., Pawlik R. T., Gualerzi C. The topographical localization of IF3 on Escherichia coli 30 S ribosomal subunits as a clue to its way of functioning. FEBS Lett. 1982 Jan 25;137(2):163–167. doi: 10.1016/0014-5793(82)80339-6. [DOI] [PubMed] [Google Scholar]
  19. Rheinberger H. J., Geigenmüller U., Wedde M., Nierhaus K. H. Parameters for the preparation of Escherichia coli ribosomes and ribosomal subunits active in tRNA binding. Methods Enzymol. 1988;164:658–670. doi: 10.1016/s0076-6879(88)64076-6. [DOI] [PubMed] [Google Scholar]
  20. Schüler D., Brimacombe R. The Escherichia coli 30S ribosomal subunit; an optimized three-dimensional fit between the ribosomal proteins and the 16S RNA. EMBO J. 1988 May;7(5):1509–1513. doi: 10.1002/j.1460-2075.1988.tb02970.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Steiner G., Kuechler E., Barta A. Photo-affinity labelling at the peptidyl transferase centre reveals two different positions for the A- and P-sites in domain V of 23S rRNA. EMBO J. 1988 Dec 1;7(12):3949–3955. doi: 10.1002/j.1460-2075.1988.tb03281.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stern S., Weiser B., Noller H. F. Model for the three-dimensional folding of 16 S ribosomal RNA. J Mol Biol. 1988 Nov 20;204(2):447–481. doi: 10.1016/0022-2836(88)90588-8. [DOI] [PubMed] [Google Scholar]
  23. Wittmann H. G. Architecture of prokaryotic ribosomes. Annu Rev Biochem. 1983;52:35–65. doi: 10.1146/annurev.bi.52.070183.000343. [DOI] [PubMed] [Google Scholar]
  24. Wower J., Hixson S. S., Zimmermann R. A. Labeling the peptidyltransferase center of the Escherichia coli ribosome with photoreactive tRNA(Phe) derivatives containing azidoadenosine at the 3' end of the acceptor arm: a model of the tRNA-ribosome complex. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5232–5236. doi: 10.1073/pnas.86.14.5232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wower J., Hixson S. S., Zimmermann R. A. Photochemical cross-linking of yeast tRNA(Phe) containing 8-azidoadenosine at positions 73 and 76 to the Escherichia coli ribosome. Biochemistry. 1988 Oct 18;27(21):8114–8121. doi: 10.1021/bi00421a021. [DOI] [PubMed] [Google Scholar]

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