Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Jul;86(14):5232–5236. doi: 10.1073/pnas.86.14.5232

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.

J Wower 1, S S Hixson 1, R A Zimmermann 1
PMCID: PMC297595  PMID: 2664777

Abstract

Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine (2N3A) at position 73 or 76 have been crosslinked to the peptidyl site of Escherichia coli ribosomes. Covalent tRNA-ribosome attachment was dependent upon the replacement of adenosine by 2N3A in the tRNA, irradiation with 300-nm light, and the presence of poly(U). In all cases, the modified tRNAs became crosslinked exclusively to 50S ribosomal subunits. While the tRNA derivative containing 2N3A at position 73 labeled only protein L27, that containing 2N3A at position 76 labeled proteins L15, L16, and L27 as well as a segment of the 23S rRNA. The site of crosslinking in the rRNA was identified as guanosine-1945, which lies within a highly conserved sequence adjacent to a number of modified bases and has not until now been identified at the peptidyltransferase center. On the basis of these results, and previously reported crosslinks from tRNA containing 8-azidoadenosine in the 3'-terminal -A-C-C-A sequence [Wower, J., Hixson, S. S. & Zimmermann, R. A. (1988) Biochemistry 27, 8114-8121], we propose a model for the arrangement of tRNA molecules at the peptidyl and aminoacyl sites that is consistent with most of the information available about the location of the peptidyltransferase center and the decoding domain of the E. coli ribosome.

Full text

PDF
5232

Images in this article

5234Image
on p.5234
5234Image
on p.5234
5235Image
on p.5235
5235Image
on p.5235
5235Image
on p.5235
5235Image
on p.5235

Selected References

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

  1. 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]
  2. Beauclerk A. A., Cundliffe E. The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli. EMBO J. 1988 Nov;7(11):3589–3594. doi: 10.1002/j.1460-2075.1988.tb03236.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Donis-Keller H. Site specific enzymatic cleavage of RNA. Nucleic Acids Res. 1979 Sep 11;7(1):179–192. doi: 10.1093/nar/7.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. Gutell R. R., Fox G. E. A compilation of large subunit RNA sequences presented in a structural format. Nucleic Acids Res. 1988;16 (Suppl):r175–r269. doi: 10.1093/nar/16.suppl.r175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hackl W., Stöffler-Meilicke M., Stöffler G. Three-dimensional location of ribosomal protein BL2 from Bacillus stearothermophilus, a key component of the peptidyl transferase center. FEBS Lett. 1988 Jun 6;233(1):119–123. doi: 10.1016/0014-5793(88)81367-x. [DOI] [PubMed] [Google Scholar]
  10. Hall C. C., Johnson D., Cooperman B. S. [3H]-p-azidopuromycin photoaffinity labeling of Escherichia coli ribosomes: evidence for site-specific interaction at U-2504 and G-2502 in domain V of 23S ribosomal RNA. Biochemistry. 1988 May 31;27(11):3983–3990. doi: 10.1021/bi00411a014. [DOI] [PubMed] [Google Scholar]
  11. Kao T., Miller D. L., Abo M., Ofengand J. Formation and properties of a covalent complex between elongation factor Tu and Phe-tRNA bearing a photoaffinity probe on its 3-(3-amino-3-carboxypropyl)uridine residue. J Mol Biol. 1983 May 25;166(3):383–405. doi: 10.1016/s0022-2836(83)80091-6. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  13. Lake J. A. Ribosome structure determined by electron microscopy of Escherichia coli small subunits, large subunits and monomeric ribosomes. J Mol Biol. 1976 Jul 25;105(1):131–139. doi: 10.1016/0022-2836(76)90200-x. [DOI] [PubMed] [Google Scholar]
  14. Lake J. A. The ribosome. Sci Am. 1981 Aug;245(2):84–97. doi: 10.1038/scientificamerican0881-84. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Lin F. L., Boublik M., Ofengand J. Immunoelectron microscopic localization of the S19 site on the 30 S ribosomal subunit which is crosslinked to A site bound transfer RNA. J Mol Biol. 1984 Jan 5;172(1):41–55. doi: 10.1016/0022-2836(84)90413-3. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Lührmann R., Bald R., Stöffler-Meilicke M., Stöffler G. Localization of the puromycin binding site on the large ribosomal subunit of Escherichia coli by immunoelectron microscopy. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7276–7280. doi: 10.1073/pnas.78.12.7276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Maly P., Brimacombe R. Refined secondary structure models for the 16S and 23S ribosomal RNA of Escherichia coli. Nucleic Acids Res. 1983 Nov 11;11(21):7263–7286. doi: 10.1093/nar/11.21.7263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Nicholson A. W., Hall C. C., Strycharz W. A., Cooperman B. S. Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analogue of puromycin. Evidence for the functional site specificity of labeling. Biochemistry. 1982 Aug 3;21(16):3809–3817. doi: 10.1021/bi00259a014. [DOI] [PubMed] [Google Scholar]
  23. Nicholson A. W., Hall C. C., Strycharz W. A., Cooperman B. S. Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analogue of puromycin. On the identification of the major covalently labeled ribosomal proteins and on the mechanism of photoincorporation. Biochemistry. 1982 Aug 3;21(16):3797–3808. doi: 10.1021/bi00259a013. [DOI] [PubMed] [Google Scholar]
  24. Nierhaus D., Nierhaus K. H. Identification of the chloramphenicol-binding protein in Escherichia coli ribosomes by partial reconstitution. Proc Natl Acad Sci U S A. 1973 Aug;70(8):2224–2228. doi: 10.1073/pnas.70.8.2224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Noller H. F. Structure of ribosomal RNA. Annu Rev Biochem. 1984;53:119–162. doi: 10.1146/annurev.bi.53.070184.001003. [DOI] [PubMed] [Google Scholar]
  26. Olson H. M., Lasater L. S., Cann P. A., Glitz D. G. Messenger RNA orientation on the ribosome. Placement by electron microscopy of antibody-complementary oligodeoxynucleotide complexes. J Biol Chem. 1988 Oct 15;263(29):15196–15204. [PubMed] [Google Scholar]
  27. Paulsen H., Robertson J. M., Wintermeyer W. Topological arrangement of two transfer RNAs on the ribosome. Fluorescence energy transfer measurements between A and P site-bound tRNAphe. J Mol Biol. 1983 Jun 25;167(2):411–426. doi: 10.1016/s0022-2836(83)80342-8. [DOI] [PubMed] [Google Scholar]
  28. Paulsen H., Wintermeyer W. Incorporation of 1,N6-ethenoadenosine into the 3' terminus of tRNA using T4 RNA ligase. 1. Preparation of yeast tRNAPhe derivatives. Eur J Biochem. 1984 Jan 2;138(1):117–123. doi: 10.1111/j.1432-1033.1984.tb07889.x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Rose S. J., 3rd, Lowary P. T., Uhlenbeck O. C. Binding of yeast tRNAPhe anticodon arm to Escherichia coli 30 S ribosomes. J Mol Biol. 1983 Jun 15;167(1):103–117. doi: 10.1016/s0022-2836(83)80036-9. [DOI] [PubMed] [Google Scholar]
  31. Schwartz I., Ofengand J. Photochemical cross-linking of unmodified acetylvalyl-tRNA to 16S RNA at the ribosomal P site. Biochemistry. 1978 Jun 27;17(13):2524–2530. doi: 10.1021/bi00606a011. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Stiege W., Glotz C., Brimacombe R. Localisation of a series of intra-RNA cross-links in the secondary and tertiary structure of 23S RNA, induced by ultraviolet irradiation of Escherichia coli 50S ribosomal subunits. Nucleic Acids Res. 1983 Mar 25;11(6):1687–1706. doi: 10.1093/nar/11.6.1687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sylvers L. A., Wower J., Hixson S. S., Zimmermann R. A. Preparation of 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for incorporation into transfer RNA. Photoaffinity labeling of Escherichia coli ribosomes. FEBS Lett. 1989 Mar 13;245(1-2):9–13. doi: 10.1016/0014-5793(89)80180-2. [DOI] [PubMed] [Google Scholar]
  36. Tejedor F., Ballesta J. P. Reaction of some macrolide antibiotics with the ribosome. Labeling of the binding site components. Biochemistry. 1986 Nov 18;25(23):7725–7731. doi: 10.1021/bi00371a066. [DOI] [PubMed] [Google Scholar]
  37. Teraoka H., Nierhaus K. H. Proteins fro Escherichia coli ribosomes involved in the binding of erythromycin. J Mol Biol. 1978 Dec 5;126(2):185–193. doi: 10.1016/0022-2836(78)90358-3. [DOI] [PubMed] [Google Scholar]
  38. Vester B., Garrett R. A. The importance of highly conserved nucleotides in the binding region of chloramphenicol at the peptidyl transfer centre of Escherichia coli 23S ribosomal RNA. EMBO J. 1988 Nov;7(11):3577–3587. doi: 10.1002/j.1460-2075.1988.tb03235.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Zweib C., Dahlberg A. E. Structural and functional analysis of Escherichia coli ribosomes containing small deletions around position 1760 in the 23S ribosomal RNA. Nucleic Acids Res. 1984 Sep 25;12(18):7135–7152. doi: 10.1093/nar/12.18.7135. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES