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. 2000 Oct;6(10):1347–1355. doi: 10.1017/s1355838200000911

The crystal structure of HIV reverse-transcription primer tRNA(Lys,3) shows a canonical anticodon loop.

P Bénas 1, G Bec 1, G Keith 1, R Marquet 1, C Ehresmann 1, B Ehresmann 1, P Dumas 1
PMCID: PMC1370007  PMID: 11073212

Abstract

We have solved to 3.3 A resolution the crystal structure of the HIV reverse-transcription primer tRNA(Lys,3). The overall structure is exactly comparable to the well-known L-shape structure first revealed by yeast tRNA(Phe). In particular, it unambiguously shows a canonical anticodon loop. This contradicts previous results in short RNA fragment studies and leads us to conclude that neither frameshifting specificities of tRNA(Lys) nor tRNA(Lys,3) primer selection by HIV are due to a specific three-dimensional anticodon structure. Comparison of our structure with the results of an NMR study on a hairpin representing a nonmodified anticodon stem-loop makes plausible the conclusion that chemical modifications of the wobble base U34 to 5-methoxycarbonyl-methyl-2-thiouridine and of A37 to 2-methylthio-N-6-threonylcarbamoyl-adenosine would be responsible for a canonical 7-nt anticodon-loop structure, whereas the unmodified form would result in a noncanonical UUU short triloop. The hexagonal crystal packing is remarkable and shows tight dimers of tRNAs forming a right-handed double superhelix. Within the dimers, the tRNAs are associated head-to-tail such that the CCA end of one tRNA interacts with the anticodon of the symmetry-related tRNA. This provides us with a partial view of a codon-anticodon interaction and gives insights into the positioning of residue 37, and of its posttranscriptional modifications, relative to the first base of the codon.

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Selected References

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  1. Agris P. F., Guenther R., Ingram P. C., Basti M. M., Stuart J. W., Sochacka E., Malkiewicz A. Unconventional structure of tRNA(Lys)SUU anticodon explains tRNA's role in bacterial and mammalian ribosomal frameshifting and primer selection by HIV-1. RNA. 1997 Apr;3(4):420–428. [PMC free article] [PubMed] [Google Scholar]
  2. Ashraf S. S., Sochacka E., Cain R., Guenther R., Malkiewicz A., Agris P. F. Single atom modification (O-->S) of tRNA confers ribosome binding. RNA. 1999 Feb;5(2):188–194. doi: 10.1017/s1355838299981529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Auffinger P., Westhof E. Singly and bifurcated hydrogen-bonded base-pairs in tRNA anticodon hairpins and ribozymes. J Mol Biol. 1999 Sep 24;292(3):467–483. doi: 10.1006/jmbi.1999.3080. [DOI] [PubMed] [Google Scholar]
  4. Brünger A. T., Adams P. D., Clore G. M., DeLano W. L., Gros P., Grosse-Kunstleve R. W., Jiang J. S., Kuszewski J., Nilges M., Pannu N. S. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905–921. doi: 10.1107/s0907444998003254. [DOI] [PubMed] [Google Scholar]
  5. Cate J. H., Yusupov M. M., Yusupova G. Z., Earnest T. N., Noller H. F. X-ray crystal structures of 70S ribosome functional complexes. Science. 1999 Sep 24;285(5436):2095–2104. doi: 10.1126/science.285.5436.2095. [DOI] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Clark B. F., Nyborg J. The ternary complex of EF-Tu and its role in protein biosynthesis. Curr Opin Struct Biol. 1997 Feb;7(1):110–116. doi: 10.1016/s0959-440x(97)80014-0. [DOI] [PubMed] [Google Scholar]
  8. Dumas P., Ennifar E., Walter P. Detection and treatment of twinning: an improvement and new results. Acta Crystallogr D Biol Crystallogr. 1999 Jun;55(Pt 6):1179–1187. doi: 10.1107/s0907444999004758. [DOI] [PubMed] [Google Scholar]
  9. Durant P. C., Davis D. R. Stabilization of the anticodon stem-loop of tRNALys,3 by an A+-C base-pair and by pseudouridine. J Mol Biol. 1999 Jan 8;285(1):115–131. doi: 10.1006/jmbi.1998.2297. [DOI] [PubMed] [Google Scholar]
  10. Fournier M., Dorizzi M., Sarger C., Labouresse J. Purification of tRNATrp, tRNAVal, and partial purification of tRNAIle and tRNAMfet from beef liver. Biochimie. 1976;58(10):1159–1165. doi: 10.1016/s0300-9084(76)80114-9. [DOI] [PubMed] [Google Scholar]
  11. Grosjean H. J., de Henau S., Crothers D. M. On the physical basis for ambiguity in genetic coding interactions. Proc Natl Acad Sci U S A. 1978 Feb;75(2):610–614. doi: 10.1073/pnas.75.2.610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Houssier C., Degée P., Nicoghosian K., Grosjean H. Effect of uridine dethiolation in the anticodon triplet of tRNA(Glu) on its association with tRNA(Phe). J Biomol Struct Dyn. 1988 Jun;5(6):1259–1266. doi: 10.1080/07391102.1988.10506468. [DOI] [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. Isel C., Lanchy J. M., Le Grice S. F., Ehresmann C., Ehresmann B., Marquet R. Specific initiation and switch to elongation of human immunodeficiency virus type 1 reverse transcription require the post-transcriptional modifications of primer tRNA3Lys. EMBO J. 1996 Feb 15;15(4):917–924. [PMC free article] [PubMed] [Google Scholar]
  15. Isel C., Marquet R., Keith G., Ehresmann C., Ehresmann B. Modified nucleotides of tRNA(3Lys) modulate primer/template loop-loop interaction in the initiation complex of HIV-1 reverse transcription. J Biol Chem. 1993 Dec 5;268(34):25269–25272. [PubMed] [Google Scholar]
  16. Isel C., Westhof E., Massire C., Le Grice S. F., Ehresmann B., Ehresmann C., Marquet R. Structural basis for the specificity of the initiation of HIV-1 reverse transcription. EMBO J. 1999 Feb 15;18(4):1038–1048. doi: 10.1093/emboj/18.4.1038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jack A., Ladner J. E., Klug A. Crystallographic refinement of yeast phenylalanine transfer RNA at 2-5A resolution. J Mol Biol. 1976 Dec 25;108(4):619–649. doi: 10.1016/s0022-2836(76)80109-x. [DOI] [PubMed] [Google Scholar]
  18. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  19. Keith G., Pixa G., Fix C., Dirheimer G. Primary structure of three tRNAs from brewer's yeast: tRNAPro2, tRNAHis1 and tRNAHis2. Biochimie. 1983 Nov-Dec;65(11-12):661–672. doi: 10.1016/s0300-9084(84)80030-9. [DOI] [PubMed] [Google Scholar]
  20. Kim S. H., Quigley G. J., Suddath F. L., McPherson A., Sneden D., Kim J. J., Weinzierl J., Rich A. Three-dimensional structure of yeast phenylalanine transfer RNA: folding of the polynucleotide chain. Science. 1973 Jan 19;179(4070):285–288. doi: 10.1126/science.179.4070.285. [DOI] [PubMed] [Google Scholar]
  21. Kumar R. K., Davis D. R. Synthesis and studies on the effect of 2-thiouridine and 4-thiouridine on sugar conformation and RNA duplex stability. Nucleic Acids Res. 1997 Mar 15;25(6):1272–1280. doi: 10.1093/nar/25.6.1272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Marquet R., Isel C., Ehresmann C., Ehresmann B. tRNAs as primer of reverse transcriptases. Biochimie. 1995;77(1-2):113–124. doi: 10.1016/0300-9084(96)88114-4. [DOI] [PubMed] [Google Scholar]
  23. Moras D., Dock A. C., Dumas P., Westhof E., Romby P., Ebel J. P., Giegé R. Anticodon-anticodon interaction induces conformational changes in tRNA: yeast tRNAAsp, a model for tRNA-mRNA recognition. Proc Natl Acad Sci U S A. 1986 Feb;83(4):932–936. doi: 10.1073/pnas.83.4.932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Parthasarathy R., Ohrt J. M., Chheda G. B. Conformation of N-(purin-6ylcarbamoyl) glycine, a hypermodified base in tRNA. Biochem Biophys Res Commun. 1974 Apr 8;57(3):649–653. doi: 10.1016/0006-291x(74)90595-6. [DOI] [PubMed] [Google Scholar]
  25. Parthasarathy R., Ohrt J. M., Chheda G. B. Modified nucleosides and conformation of anticodon loops: crystal structure of t6A and g6A. Biochemistry. 1977 Nov 15;16(23):4999–5008. doi: 10.1021/bi00642a010. [DOI] [PubMed] [Google Scholar]
  26. Puglisi E. V., Puglisi J. D. HIV-1 A-rich RNA loop mimics the tRNA anticodon structure. Nat Struct Biol. 1998 Dec;5(12):1033–1036. doi: 10.1038/4141. [DOI] [PubMed] [Google Scholar]
  27. Quigley G. J., Rich A. Structural domains of transfer RNA molecules. Science. 1976 Nov 19;194(4267):796–806. doi: 10.1126/science.790568. [DOI] [PubMed] [Google Scholar]
  28. Raba M., Limburg K., Burghagen M., Katze J. R., Simsek M., Heckman J. E., Rajbhandary U. L., Gross H. J. Nucleotide sequence of three isoaccepting lysine tRNAs from rabbit liver and SV40-transformed mouse fibroblasts. Eur J Biochem. 1979 Jun;97(1):305–318. doi: 10.1111/j.1432-1033.1979.tb13115.x. [DOI] [PubMed] [Google Scholar]
  29. Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
  30. Rhee S., Han Z. j., Liu K., Miles H. T., Davies D. R. Structure of a triple helical DNA with a triplex-duplex junction. Biochemistry. 1999 Dec 21;38(51):16810–16815. doi: 10.1021/bi991811m. [DOI] [PubMed] [Google Scholar]
  31. Robertus J. D., Ladner J. E., Finch J. T., Rhodes D., Brown R. S., Clark B. F., Klug A. Structure of yeast phenylalanine tRNA at 3 A resolution. Nature. 1974 Aug 16;250(467):546–551. doi: 10.1038/250546a0. [DOI] [PubMed] [Google Scholar]
  32. Stark H., Rodnina M. V., Rinke-Appel J., Brimacombe R., Wintermeyer W., van Heel M. Visualization of elongation factor Tu on the Escherichia coli ribosome. Nature. 1997 Sep 25;389(6649):403–406. doi: 10.1038/38770. [DOI] [PubMed] [Google Scholar]
  33. Tsuchihashi Z., Brown P. O. Sequence requirements for efficient translational frameshifting in the Escherichia coli dnaX gene and the role of an unstable interaction between tRNA(Lys) and an AAG lysine codon. Genes Dev. 1992 Mar;6(3):511–519. doi: 10.1101/gad.6.3.511. [DOI] [PubMed] [Google Scholar]
  34. Watanabe K., Hayashi N., Oyama A., Nishikawa K., Ueda T., Miura K. Unusual anticodon loop structure found in E.coli lysine tRNA. Nucleic Acids Res. 1994 Jan 11;22(1):79–87. doi: 10.1093/nar/22.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Watanabe K., Yokoyama S., Hansske F., Kasai H., Miyazawa T. CD and NMR studies on the conformational thermostability of 2-thioribothymidine found in the T psi C loop of thermophile tRNA. Biochem Biophys Res Commun. 1979 Nov 28;91(2):671–677. doi: 10.1016/0006-291x(79)91574-2. [DOI] [PubMed] [Google Scholar]
  36. Westhof E., Dumas P., Moras D. Crystallographic refinement of yeast aspartic acid transfer RNA. J Mol Biol. 1985 Jul 5;184(1):119–145. doi: 10.1016/0022-2836(85)90048-8. [DOI] [PubMed] [Google Scholar]
  37. Yeates T. O. Detecting and overcoming crystal twinning. Methods Enzymol. 1997;276:344–358. [PubMed] [Google Scholar]
  38. Yokoyama S., Watanabe T., Murao K., Ishikura H., Yamaizumi Z., Nishimura S., Miyazawa T. Molecular mechanism of codon recognition by tRNA species with modified uridine in the first position of the anticodon. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4905–4909. doi: 10.1073/pnas.82.15.4905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yokoyama S., Yamaizumi Z., Nishimura S., Miyazawa T. 1H NMR studies on the conformational characteristics of 2-thiopyrimidine nucleotides found in transfer RNAs. Nucleic Acids Res. 1979 Jun 11;6(7):2611–2626. doi: 10.1093/nar/6.7.2611. [DOI] [PMC free article] [PubMed] [Google Scholar]

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