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. 1996 Feb 1;24(3):441–449. doi: 10.1093/nar/24.3.441

Extended interactions between the primer tRNAi(Met) and genomic RNA of the yeast Ty1 retrotransposon.

S Friant 1, T Heyman 1, M L Wilhelm 1, F X Wilhelm 1
PMCID: PMC145666  PMID: 8602356

Abstract

Reverse transcription of the yeast Ty1 retrotransposon is primed by tRNAi(Met) base paired to the primer binding site near the 5'-end of Ty1 genomic RNA. To understand the molecular basis of the tRNAi(Met)-Ty1 RNA interaction the secondary structure of the binary complex was analysed. Enzymatic probes were used to test the conformation of tRNAi(Met) and of Ty1 RNA in the free form and in the complex. A secondary structure model of the tRNAi(Met) Ty1 RNA complex consistent with the probing data was constructed with the help of a computer program. The model shows that besides interactions between the primer binding site and the last 10 nt at the 3'-end of tRNAi(Met), three short regions of Ty1 RNA named boxes 0, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Mutations were made in the boxes or in the complementary sequences of tRNAi(Met) to study the contribution of these sequences to formation of the complex. We find that interaction with at least one of the two boxes 0 or 1 is absolutely required for efficient annealing of the two RNAs. Sequence comparison showing that the primary sequence of the boxes is strictly conserved in Ty1 and Ty2 elements and previously published in vivo results underline the functional importance of the primary sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primary tRNAi(Met) play a role in the reverse transcription pathway.

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Selected References

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  1. Aiyar A., Cobrinik D., Ge Z., Kung H. J., Leis J. Interaction between retroviral U5 RNA and the T psi C loop of the tRNA(Trp) primer is required for efficient initiation of reverse transcription. J Virol. 1992 Apr;66(4):2464–2472. doi: 10.1128/jvi.66.4.2464-2472.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aiyar A., Ge Z., Leis J. A specific orientation of RNA secondary structures is required for initiation of reverse transcription. J Virol. 1994 Feb;68(2):611–618. doi: 10.1128/jvi.68.2.611-618.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barat C., Lullien V., Schatz O., Keith G., Nugeyre M. T., Grüninger-Leitch F., Barré-Sinoussi F., LeGrice S. F., Darlix J. L. HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO J. 1989 Nov;8(11):3279–3285. doi: 10.1002/j.1460-2075.1989.tb08488.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barat C., Schatz O., Le Grice S., Darlix J. L. Analysis of the interactions of HIV1 replication primer tRNA(Lys,3) with nucleocapsid protein and reverse transcriptase. J Mol Biol. 1993 May 20;231(2):185–190. doi: 10.1006/jmbi.1993.1273. [DOI] [PubMed] [Google Scholar]
  5. Boeke J. D., Eichinger D., Castrillon D., Fink G. R. The Saccharomyces cerevisiae genome contains functional and nonfunctional copies of transposon Ty1. Mol Cell Biol. 1988 Apr;8(4):1432–1442. doi: 10.1128/mcb.8.4.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boeke J. D., Garfinkel D. J., Styles C. A., Fink G. R. Ty elements transpose through an RNA intermediate. Cell. 1985 Mar;40(3):491–500. doi: 10.1016/0092-8674(85)90197-7. [DOI] [PubMed] [Google Scholar]
  7. Chapman K. B., Byström A. S., Boeke J. D. Initiator methionine tRNA is essential for Ty1 transposition in yeast. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3236–3240. doi: 10.1073/pnas.89.8.3236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clare J., Farabaugh P. Nucleotide sequence of a yeast Ty element: evidence for an unusual mechanism of gene expression. Proc Natl Acad Sci U S A. 1985 May;82(9):2829–2833. doi: 10.1073/pnas.82.9.2829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cordell B., Swanstrom R., Goodman H. M., Bishop J. M. tRNATrp as primer for RNA-directed DNA polymerase: structural determinants of function. J Biol Chem. 1979 Mar 25;254(6):1866–1874. [PubMed] [Google Scholar]
  10. Davanloo P., Rosenberg A. H., Dunn J. J., Studier F. W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2035–2039. doi: 10.1073/pnas.81.7.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Despons L., Senger B., Fasiolo F., Walter P. Binding of the yeast tRNA(Met) anticodon by the cognate methionyl-tRNA synthetase involves at least two independent peptide regions. J Mol Biol. 1992 Jun 5;225(3):897–907. doi: 10.1016/0022-2836(92)90409-d. [DOI] [PubMed] [Google Scholar]
  12. Ehresmann C., Baudin F., Mougel M., Romby P., Ebel J. P., Ehresmann B. Probing the structure of RNAs in solution. Nucleic Acids Res. 1987 Nov 25;15(22):9109–9128. doi: 10.1093/nar/15.22.9109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Isel C., Ehresmann C., Keith G., Ehresmann B., Marquet R. Initiation of reverse transcription of HIV-1: secondary structure of the HIV-1 RNA/tRNA(3Lys) (template/primer). J Mol Biol. 1995 Mar 24;247(2):236–250. doi: 10.1006/jmbi.1994.0136. [DOI] [PubMed] [Google Scholar]
  14. Jaeger J. A., Turner D. H., Zuker M. Predicting optimal and suboptimal secondary structure for RNA. Methods Enzymol. 1990;183:281–306. doi: 10.1016/0076-6879(90)83019-6. [DOI] [PubMed] [Google Scholar]
  15. Keeney J. B., Chapman K. B., Lauermann V., Voytas D. F., Aström S. U., von Pawel-Rammingen U., Byström A., Boeke J. D. Multiple molecular determinants for retrotransposition in a primer tRNA. Mol Cell Biol. 1995 Jan;15(1):217–226. doi: 10.1128/mcb.15.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Khan R., Giedroc D. P. Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA. J Biol Chem. 1992 Apr 5;267(10):6689–6695. [PubMed] [Google Scholar]
  17. Kohlstaedt L. A., Steitz T. A. Reverse transcriptase of human immunodeficiency virus can use either human tRNA(3Lys) or Escherichia coli tRNA(2Gln) as a primer in an in vitro primer-utilization assay. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9652–9656. doi: 10.1073/pnas.89.20.9652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krol A., Carbon P. A guide for probing native small nuclear RNA and ribonucleoprotein structures. Methods Enzymol. 1989;180:212–227. doi: 10.1016/0076-6879(89)80103-x. [DOI] [PubMed] [Google Scholar]
  19. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Peattie D. A., Gilbert W. Chemical probes for higher-order structure in RNA. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4679–4682. doi: 10.1073/pnas.77.8.4679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pochart P., Agoutin B., Fix C., Keith G., Heyman T. A very poorly expressed tRNA(Ser) is highly concentrated together with replication primer initiator tRNA(Met) in the yeast Ty1 virus-like particles. Nucleic Acids Res. 1993 Apr 11;21(7):1517–1521. doi: 10.1093/nar/21.7.1517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stucka R., Lochmüller H., Feldmann H. Ty4, a novel low-copy number element in Saccharomyces cerevisiae: one copy is located in a cluster of Ty elements and tRNA genes. Nucleic Acids Res. 1989 Jul 11;17(13):4993–5001. doi: 10.1093/nar/17.13.4993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Varmus H. Retroviruses. Science. 1988 Jun 10;240(4858):1427–1435. doi: 10.1126/science.3287617. [DOI] [PubMed] [Google Scholar]
  24. Warmington J. R., Waring R. B., Newlon C. S., Indge K. J., Oliver S. G. Nucleotide sequence characterization of Ty 1-17, a class II transposon from yeast. Nucleic Acids Res. 1985 Sep 25;13(18):6679–6693. doi: 10.1093/nar/13.18.6679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Warmington J. R., Waring R. B., Newlon C. S., Indge K. J., Oliver S. G. Nucleotide sequence characterization of Ty 1-17, a class II transposon from yeast. Nucleic Acids Res. 1985 Sep 25;13(18):6679–6693. doi: 10.1093/nar/13.18.6679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wilhelm M., Wilhelm F. X., Keith G., Agoutin B., Heyman T. Yeast Ty1 retrotransposon: the minus-strand primer binding site and a cis-acting domain of the Ty1 RNA are both important for packaging of primer tRNA inside virus-like particles. Nucleic Acids Res. 1994 Nov 11;22(22):4560–4565. doi: 10.1093/nar/22.22.4560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wimberly B., Varani G., Tinoco I., Jr The conformation of loop E of eukaryotic 5S ribosomal RNA. Biochemistry. 1993 Feb 2;32(4):1078–1087. doi: 10.1021/bi00055a013. [DOI] [PubMed] [Google Scholar]
  28. Zuker M. On finding all suboptimal foldings of an RNA molecule. Science. 1989 Apr 7;244(4900):48–52. doi: 10.1126/science.2468181. [DOI] [PubMed] [Google Scholar]

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