Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1993 Feb;12(2):617–623. doi: 10.1002/j.1460-2075.1993.tb05694.x

Topography of the E site on the Escherichia coli ribosome.

J Wower 1, P Scheffer 1, L A Sylvers 1, W Wintermeyer 1, R A Zimmermann 1
PMCID: PMC413245  PMID: 8440251

Abstract

Three photoreactive tRNA probes have been utilized in order to identify ribosomal components that are in contact with the aminoacyl acceptor end and the anticodon loop of tRNA bound to the E site of Escherichia coli ribosomes. Two of the probes were derivatives of E. coli tRNA(Phe) in which adenosines at positions 73 and 76 were replaced by 2-azidoadenosine. The third probe was derived from yeast tRNA(Phe) by substituting wyosine at position 37 with 2-azidoadenosine. Despite the modifications, all of the photoreactive tRNA species were able to bind to the E site of E. coli ribosomes programmed with poly(A) and, upon irradiation, formed covalent adducts with the ribosomal subunits. The tRNA(Phe) probes modified at or near the 3' terminus exclusively labeled protein L33 in the 50S subunit. The tRNA(Phe) derivative containing 2-azidoadenosine within the anticodon loop became cross-linked to protein S11 as well as to a segment of the 16S rRNA encompassing the 3'-terminal 30 nucleotides. We have located the two extremities of the E site-bound tRNA on the ribosomal subunits according to the positions of L33, S11 and the 3' end of 16S rRNA defined by immune electron microscopy. Our results demonstrate conclusively that the E site is topographically distinct from either the P site or the A site, and that it is located alongside the P site as expected for the tRNA exit site.

Full text

PDF
617

Images in this article

619Image
on p.619
620Image
on p.620
621Image
on p.621
621Image
on p.621

Selected References

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

  1. Branlant C., Krol A., Machatt A., Ebel J. P. The secondary structure of the protein L1 binding region of ribosomal 23S RNA. Homologies with putative secondary structures of the L11 mRNA and of a region of mitochondrial 16S rRNA. Nucleic Acids Res. 1981 Jan 24;9(2):293–307. doi: 10.1093/nar/9.2.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cooperman B. S. Affinity labeling of ribosomes. Methods Enzymol. 1988;164:341–361. doi: 10.1016/s0076-6879(88)64054-7. [DOI] [PubMed] [Google Scholar]
  3. Geigenmüller U., Nierhaus K. H. Significance of the third tRNA binding site, the E site, on E. coli ribosomes for the accuracy of translation: an occupied E site prevents the binding of non-cognate aminoacyl-tRNA to the A site. EMBO J. 1990 Dec;9(13):4527–4533. doi: 10.1002/j.1460-2075.1990.tb07904.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Grajevskaja R. A., Ivanov Y. V., Saminsky E. M. 70-S ribosomes of Escherichia coli have an additional site for deacylated tRNA binding. Eur J Biochem. 1982 Nov;128(1):47–52. doi: 10.1111/j.1432-1033.1982.tb06929.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Hayase Y., Inoue H., Ohtsuka E. Secondary structure in formylmethionine tRNA influences the site-directed cleavage of ribonuclease H using chimeric 2'-O-methyl oligodeoxyribonucleotides. Biochemistry. 1990 Sep 18;29(37):8793–8797. doi: 10.1021/bi00489a041. [DOI] [PubMed] [Google Scholar]
  8. Helser T. L., Davies J. E., Dahlberg J. E. Change in methylation of 16S ribosomal RNA associated with mutation to kasugamycin resistance in Escherichia coli. Nat New Biol. 1971 Sep 1;233(35):12–14. doi: 10.1038/newbio233012a0. [DOI] [PubMed] [Google Scholar]
  9. Kirillov S. V., Makarov E. M., Semenkov YuP Quantitative study of interaction of deacylated tRNA with Escherichia coli ribosomes. Role of 50 S subunits in formation of the E site. FEBS Lett. 1983 Jun 27;157(1):91–94. doi: 10.1016/0014-5793(83)81122-3. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Lake J. A., Strycharz W. A. Ribosomal proteins L1, L17 and L27 from Escherichia coli localized at single sites on the large subunit by immune electron microscopy. J Mol Biol. 1981 Dec 25;153(4):979–992. doi: 10.1016/0022-2836(81)90462-9. [DOI] [PubMed] [Google Scholar]
  12. Lill R., Lepier A., Schwägele F., Sprinzl M., Vogt H., Wintermeyer W. Specific recognition of the 3'-terminal adenosine of tRNAPhe in the exit site of Escherichia coli ribosomes. J Mol Biol. 1988 Oct 5;203(3):699–705. doi: 10.1016/0022-2836(88)90203-3. [DOI] [PubMed] [Google Scholar]
  13. Lill R., Robertson J. M., Wintermeyer W. Affinities of tRNA binding sites of ribosomes from Escherichia coli. Biochemistry. 1986 Jun 3;25(11):3245–3255. doi: 10.1021/bi00359a025. [DOI] [PubMed] [Google Scholar]
  14. Lill R., Robertson J. M., Wintermeyer W. Binding of the 3' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation. EMBO J. 1989 Dec 1;8(12):3933–3938. doi: 10.1002/j.1460-2075.1989.tb08574.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lill R., Robertson J. M., Wintermeyer W. tRNA binding sites of ribosomes from Escherichia coli. Biochemistry. 1984 Dec 18;23(26):6710–6717. doi: 10.1021/bi00321a066. [DOI] [PubMed] [Google Scholar]
  16. Lill R., Wintermeyer W. Destabilization of codon-anticodon interaction in the ribosomal exit site. J Mol Biol. 1987 Jul 5;196(1):137–148. doi: 10.1016/0022-2836(87)90516-x. [DOI] [PubMed] [Google Scholar]
  17. Lill R., Wintermeyer W. Quantitative indicator assay of tRNA binding to the ribosomal P and A sites. Methods Enzymol. 1988;164:597–611. doi: 10.1016/s0076-6879(88)64072-9. [DOI] [PubMed] [Google Scholar]
  18. Lim V., Venclovas C., Spirin A., Brimacombe R., Mitchell P., Müller F. How are tRNAs and mRNA arranged in the ribosome? An attempt to correlate the stereochemistry of the tRNA-mRNA interaction with constraints imposed by the ribosomal topography. Nucleic Acids Res. 1992 Jun 11;20(11):2627–2637. doi: 10.1093/nar/20.11.2627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  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. Intermediate states in the movement of transfer RNA in the ribosome. Nature. 1989 Nov 9;342(6246):142–148. doi: 10.1038/342142a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Olson H. M., Glitz D. G. Ribosome structure: localization of 3' end of RNA in small subunit by immunoelectronmicroscopy. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3769–3773. doi: 10.1073/pnas.76.8.3769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Paulsen H., Wintermeyer W. tRNA topography during translocation: steady-state and kinetic fluorescence energy-transfer studies. Biochemistry. 1986 May 20;25(10):2749–2756. doi: 10.1021/bi00358a002. [DOI] [PubMed] [Google Scholar]
  26. Politz S. M., Glitz D. G. Ribosome structure: localization of N6,N6-dimethyladenosine by electron microscopy of a ribosome-antibody complex. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1468–1472. doi: 10.1073/pnas.74.4.1468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Redl B., Walleczek J., Stöffler-Meilicke M., Stöffler G. Immunoblotting analysis of protein-protein crosslinks within the 50S ribosomal subunit of Escherichia coli. A study using dimethylsuberimidate as crosslinking reagent. Eur J Biochem. 1989 May 1;181(2):351–356. doi: 10.1111/j.1432-1033.1989.tb14731.x. [DOI] [PubMed] [Google Scholar]
  29. Rheinberger H. J., Sternbach H., Nierhaus K. H. Three tRNA binding sites on Escherichia coli ribosomes. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5310–5314. doi: 10.1073/pnas.78.9.5310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Robertson J. M., Paulsen H., Wintermeyer W. Pre-steady-state kinetics of ribosomal translocation. J Mol Biol. 1986 Nov 20;192(2):351–360. doi: 10.1016/0022-2836(86)90370-0. [DOI] [PubMed] [Google Scholar]
  31. Robertson J. M., Wintermeyer W. Mechanism of ribosomal translocation. tRNA binds transiently to an exit site before leaving the ribosome during translocation. J Mol Biol. 1987 Aug 5;196(3):525–540. doi: 10.1016/0022-2836(87)90030-1. [DOI] [PubMed] [Google Scholar]
  32. Schwartz I., Ofengand J. Photo-affinity labeling of tRNA binding sites in macromolecules. I. Linking of the phenacyl-p-azide of 4-thiouridine in (Escherichia coli) valyl-tRNA to 16S RNA at the ribosomal P site. Proc Natl Acad Sci U S A. 1974 Oct;71(10):3951–3955. doi: 10.1073/pnas.71.10.3951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shatsky I. N., Mochalova L. V., Kojouharova M. S., Bogdanov A. A., Vasiliev V. D. Localization of the 3' end of Escherichia coli 16 S RNA by electron microscopy of antibody-labelled subunits. J Mol Biol. 1979 Oct 9;133(4):501–515. doi: 10.1016/0022-2836(79)90404-2. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Steitz J. A., Jakes K. How ribosomes select initiator regions in mRNA: base pair formation between the 3' terminus of 16S rRNA and the mRNA during initiation of protein synthesis in Escherichia coli. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4734–4738. doi: 10.1073/pnas.72.12.4734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sylvers L. A., Kopylov A. M., Wower J., Hixson S. S., Zimmermann R. A. Photochemical cross-linking of the anticodon loop of yeast tRNA(Phe) to 30S-subunit protein S7 at the ribosomal A and P sites. Biochimie. 1992 Apr;74(4):381–389. doi: 10.1016/0300-9084(92)90116-v. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Walleczek J., Martin T., Redl B., Stöffler-Meilicke M., Stöffler G. Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli. Biochemistry. 1989 May 2;28(9):4099–4105. doi: 10.1021/bi00435a071. [DOI] [PubMed] [Google Scholar]
  39. Walleczek J., Redl B., Stöffler-Meilicke M., Stöffler G. Protein-protein cross-linking of the 50 S ribosomal subunit of Escherichia coli using 2-iminothiolane. Identification of cross-links by immunoblotting techniques. J Biol Chem. 1989 Mar 5;264(7):4231–4237. [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Wower J., Zimmermann R. A. A consonant model of the tRNA-ribosome complex during the elongation cycle of translation. Biochimie. 1991 Jul-Aug;73(7-8):961–969. doi: 10.1016/0300-9084(91)90137-p. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES