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
. 1992 Sep 1;89(17):7851–7855. doi: 10.1073/pnas.89.17.7851

Hydroxyl radical cleavage of tRNA in the ribosomal P site.

A Hüttenhofer 1, H F Noller 1
PMCID: PMC49812  PMID: 1381501

Abstract

Hydroxyl radical is a useful probe of the accessibility of the sugar moiety of nucleic acids to solvent. Here we compare the accessibility of free and ribosome-bound yeast tRNA(Phe), Escherichia coli tRNA(Phe), and E. coli tRNA(Leu2) to attack by hydroxyl radicals generated from Fe(2+)-EDTA. When bound to the P site of 30S ribosomal subunits, a discrete region, corresponding almost precisely to the anticodon stem-loop, is strongly protected; weaker protection is observed in the 3' strand of the D stem and in the variable loop. The protected nucleotides constitute a well-defined substructure, corresponding to the lower half of the anticodon-D loop coaxial arm of the tRNA crystal structure. This result suggests that the 30S P site contains a pocket that becomes inaccessible to the Fe(2+)-EDTA complex when tRNA is bound, whose minimum dimensions can be inferred from the boundaries of the protected region of tRNA. When bound to the P site of 70S ribosomes, the entire tRNA backbone becomes inaccessible to hydroxyl radicals. Since previous studies have shown that virtually the entire footprint of a P-site tRNA on 16S and 23S rRNAs is mimicked by the extremities of the tRNA (the anticodon stem-loop plus the 3'-terminal aminoacyl-pentanucleotide), protection of the entire tRNA was unexpected. We conclude that protection of the elbow of tRNA is due either to interactions with ribosomal proteins or to enclosure in an inaccessible site formed by association of the two ribosomal subunits.

Full text

PDF
7851

Images in this article

7852Image
on p.7852
7853Image
on p.7853
7854Image
on p.7854
7854Image
on p.7854

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. Bertram S., Göringer U., Wagner R. Structural investigation of Phe-tRNAPhe from E.coli bound to the ribosomal A-site. Nucleic Acids Res. 1983 Feb 11;11(3):575–589. doi: 10.1093/nar/11.3.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Björk G. R., Ericson J. U., Gustafsson C. E., Hagervall T. G., Jönsson Y. H., Wikström P. M. Transfer RNA modification. Annu Rev Biochem. 1987;56:263–287. doi: 10.1146/annurev.bi.56.070187.001403. [DOI] [PubMed] [Google Scholar]
  4. Caskey C. T., Beaudet A. L., Scolnick E. M., Rosman M. Hydrolysis of fMet-tRNA by peptidyl transferase. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3163–3167. doi: 10.1073/pnas.68.12.3163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Donis-Keller H., Maxam A. M., Gilbert W. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977 Aug;4(8):2527–2538. doi: 10.1093/nar/4.8.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Douthwaite S., Garrett R. A., Wagner R. Comparison of Escherichia coli tRNAPhe in the free state, in the ternary complex and in the ribosomal A and P sites by chemical probing. Eur J Biochem. 1983 Mar 15;131(2):261–269. doi: 10.1111/j.1432-1033.1983.tb07258.x. [DOI] [PubMed] [Google Scholar]
  7. England T. E., Bruce A. G., Uhlenbeck O. C. Specific labeling of 3' termini of RNA with T4 RNA ligase. Methods Enzymol. 1980;65(1):65–74. doi: 10.1016/s0076-6879(80)65011-3. [DOI] [PubMed] [Google Scholar]
  8. Farber N., Cantor C. R. Comparison of the structures of free and ribosome-bound tRNAPhe by using slow tritium exchange. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5135–5139. doi: 10.1073/pnas.77.9.5135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hall K. B., Sampson J. R., Uhlenbeck O. C., Redfield A. G. Structure of an unmodified tRNA molecule. Biochemistry. 1989 Jul 11;28(14):5794–5801. doi: 10.1021/bi00440a014. [DOI] [PubMed] [Google Scholar]
  10. Latham J. A., Cech T. R. Defining the inside and outside of a catalytic RNA molecule. Science. 1989 Jul 21;245(4915):276–282. doi: 10.1126/science.2501870. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. Moazed D., Noller H. F. Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3725–3728. doi: 10.1073/pnas.88.9.3725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Moazed D., Stern S., Noller H. F. Rapid chemical probing of conformation in 16 S ribosomal RNA and 30 S ribosomal subunits using primer extension. J Mol Biol. 1986 Feb 5;187(3):399–416. doi: 10.1016/0022-2836(86)90441-9. [DOI] [PubMed] [Google Scholar]
  18. Peattie D. A., Herr W. Chemical probing of the tRNA--ribosome complex. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2273–2277. doi: 10.1073/pnas.78.4.2273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Tullius T. D., Dombroski B. A. Iron(II) EDTA used to measure the helical twist along any DNA molecule. Science. 1985 Nov 8;230(4726):679–681. doi: 10.1126/science.2996145. [DOI] [PubMed] [Google Scholar]
  22. Wakao H., Romby P., Westhof E., Laalami S., Grunberg-Manago M., Ebel J. P., Ehresmann C., Ehresmann B. The solution structure of the Escherichia coli initiator tRNA and its interactions with initiation factor 2 and the ribosomal 30 S subunit. J Biol Chem. 1989 Dec 5;264(34):20363–20371. [PubMed] [Google Scholar]
  23. 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]
  24. Wu J. C., Kozarich J. W., Stubbe J. The mechanism of free base formation from DNA by bleomycin. A proposal based on site specific tritium release from Poly(dA.dU). J Biol Chem. 1983 Apr 25;258(8):4694–4697. [PubMed] [Google Scholar]
  25. Zamir A., Miskin R., Elson D. Inactivation and reactivation of ribosomal subunits: amino acyl-transfer RNA binding activity of the 30 s subunit of Escherichia coli. J Mol Biol. 1971 Sep 14;60(2):347–364. doi: 10.1016/0022-2836(71)90299-3. [DOI] [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