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. 1998 Jun 15;17(12):3478–3483. doi: 10.1093/emboj/17.12.3478

EF-G-catalyzed translocation of anticodon stem-loop analogs of transfer RNA in the ribosome.

S Joseph 1, H F Noller 1
PMCID: PMC1170684  PMID: 9628883

Abstract

Translocation, catalyzed by elongation factor EF-G, is the precise movement of the tRNA-mRNA complex within the ribosome following peptide bond formation. Here we examine the structural requirement for A- and P-site tRNAs in EF-G-catalyzed translocation by substituting anticodon stem-loop (ASL) analogs for the respective tRNAs. Translocation of mRNA and tRNA was monitored independently; mRNA movement was assayed by toeprinting, while tRNA and ASL movement was monitored by hydroxyl radical probing by Fe(II) tethered to the ASLs and by chemical footprinting. Translocation depends on occupancy of both A and P sites by tRNA bound in a mRNA-dependent fashion. The requirement for an A-site tRNA can be satisfied by a 15 nucleotide ASL analog comprising only a 4 base pair (bp) stem and a 7 nucleotide anticodon loop. Translocation of the ASL is both EF-G- and GTP-dependent, and is inhibited by the translocational inhibitor thiostrepton. These findings show that the D, T and acceptor stem regions of A-site tRNA are not essential for EF-G-dependent translocation. In contrast, no translocation occurs if the P-site tRNA is substituted with an ASL, indicating that other elements of P-site tRNA structure are required for translocation. We also tested the effect of increasing the A-site ASL stem length from 4 to 33 bp on translocation from A to P site. Translocation efficiency decreases as the ASL stem extends beyond 22 bp, corresponding approximately to the maximum dimension of tRNA along the anticodon-D arm axis. This result suggests that a structural feature of the ribosome between the A and P sites, interferes with movement of tRNA analogs that exceed the normal dimensions of the coaxial tRNA anticodon-D arm.

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Selected References

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

  1. Agrawal R. K., Penczek P., Grassucci R. A., Li Y., Leith A., Nierhaus K. H., Frank J. Direct visualization of A-, P-, and E-site transfer RNAs in the Escherichia coli ribosome. Science. 1996 Feb 16;271(5251):1000–1002. doi: 10.1126/science.271.5251.1000. [DOI] [PubMed] [Google Scholar]
  2. Belitsina N. V., Tnalina G. Z., Spirin A. S. Template-free ribosomal synthesis of polylysine from lysyl-tRNA. FEBS Lett. 1981 Aug 31;131(2):289–292. doi: 10.1016/0014-5793(81)80387-0. [DOI] [PubMed] [Google Scholar]
  3. Belitsina N. V., Tnalina G. Z., Spirin A. S. Template-free ribosomal synthesis of polypeptides from aminoacyl-tRNAs. Biosystems. 1982;15(3):233–241. doi: 10.1016/0303-2647(82)90008-9. [DOI] [PubMed] [Google Scholar]
  4. Czworkowski J., Moore P. B. The elongation phase of protein synthesis. Prog Nucleic Acid Res Mol Biol. 1996;54:293–332. doi: 10.1016/s0079-6603(08)60366-9. [DOI] [PubMed] [Google Scholar]
  5. Hartz D., McPheeters D. S., Gold L. Selection of the initiator tRNA by Escherichia coli initiation factors. Genes Dev. 1989 Dec;3(12A):1899–1912. doi: 10.1101/gad.3.12a.1899. [DOI] [PubMed] [Google Scholar]
  6. Hartz D., McPheeters D. S., Traut R., Gold L. Extension inhibition analysis of translation initiation complexes. Methods Enzymol. 1988;164:419–425. doi: 10.1016/s0076-6879(88)64058-4. [DOI] [PubMed] [Google Scholar]
  7. Holschuh K., Bonin J., Gassen H. G. Mechanism of translocation: effect of cognate transfer ribonucleic acids on the binding of AUGUn to 70S ribosomes. Biochemistry. 1980 Dec 9;19(25):5857–5864. doi: 10.1021/bi00566a030. [DOI] [PubMed] [Google Scholar]
  8. Hüttenhofer A., Noller H. F. Footprinting mRNA-ribosome complexes with chemical probes. EMBO J. 1994 Aug 15;13(16):3892–3901. doi: 10.1002/j.1460-2075.1994.tb06700.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hüttenhofer A., Noller H. F. Hydroxyl radical cleavage of tRNA in the ribosomal P site. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7851–7855. doi: 10.1073/pnas.89.17.7851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ishitsuka H., Kuriki Y., Kaji A. Release of transfer ribonucleic acid from ribosomes. A G factor and guanosine triphosphate-dependent reaction. J Biol Chem. 1970 Jul 10;245(13):3346–3351. [PubMed] [Google Scholar]
  11. Joseph S., Noller H. F. Mapping the rRNA neighborhood of the acceptor end of tRNA in the ribosome. EMBO J. 1996 Feb 15;15(4):910–916. [PMC free article] [PubMed] [Google Scholar]
  12. Joseph S., Weiser B., Noller H. F. Mapping the inside of the ribosome with an RNA helical ruler. Science. 1997 Nov 7;278(5340):1093–1098. doi: 10.1126/science.278.5340.1093. [DOI] [PubMed] [Google Scholar]
  13. Kaziro Y. The role of guanosine 5'-triphosphate in polypeptide chain elongation. Biochim Biophys Acta. 1978 Sep 21;505(1):95–127. doi: 10.1016/0304-4173(78)90009-5. [DOI] [PubMed] [Google Scholar]
  14. Liljas A., AEvarsson A., al-Karadaghi S., Garber M., Zheltonosova J., Brazhnikov E. Crystallographic studies of elongation factor G. Biochem Cell Biol. 1995 Nov-Dec;73(11-12):1209–1216. doi: 10.1139/o95-130. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Lucas-Lenard J., Haenni A. L. Release of transfer RNA during peptide chain elongation. Proc Natl Acad Sci U S A. 1969 May;63(1):93–97. doi: 10.1073/pnas.63.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Modolell J., Cabrer B., Vázquez D. The stoichiometry of ribosomal translocation. J Biol Chem. 1973 Dec 25;248(24):8356–8360. [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. Roufa D. J., Skogerson L. E., Leder P. Translation of phage Qbeta mRNA: a test of the two-site model for ribosomal function. Nature. 1970 Aug 8;227(5258):567–570. doi: 10.1038/227567a0. [DOI] [PubMed] [Google Scholar]
  22. Spirin A. S. Ribosomal translocation: facts and models. Prog Nucleic Acid Res Mol Biol. 1985;32:75–114. doi: 10.1016/s0079-6603(08)60346-3. [DOI] [PubMed] [Google Scholar]
  23. Stark H., Orlova E. V., Rinke-Appel J., Jünke N., Mueller F., Rodnina M., Wintermeyer W., Brimacombe R., van Heel M. Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy. Cell. 1997 Jan 10;88(1):19–28. doi: 10.1016/s0092-8674(00)81854-1. [DOI] [PubMed] [Google Scholar]
  24. Stern S., Moazed D., Noller H. F. Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. Methods Enzymol. 1988;164:481–489. doi: 10.1016/s0076-6879(88)64064-x. [DOI] [PubMed] [Google Scholar]

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