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. 1996 Oct 15;24(20):3974–3981. doi: 10.1093/nar/24.20.3974

Structure of HIV-1 TAR RNA in the absence of ligands reveals a novel conformation of the trinucleotide bulge.

F Aboul-ela 1, J Karn 1, G Varani 1
PMCID: PMC146214  PMID: 8918800

Abstract

Efficient transcription from the human immunodeficiency virus (HIV) promoter depends on binding of the viral regulatory protein Tat to a cis-acting RNA regulatory element, TAR. Tat binds at a trinucleotide bulge located near the apex of the TAR stem-loop structure. An essential feature of Tat-TAR interaction is that the protein induces a conformational change in TAR that repositions the functional groups on the bases and the phosphate backbone that are critical for specific intermolecular recognition of TAR RNA. We have previously determined a high resolution structure for the bound form of TAR RNA using heteronuclear NMR. Here, we describe a high resolution structure of the free TAR RNA based on 871 experimentally determined restraints. In the free TAR RNA, bulged residues U23 and C24 are stacked within the helix, while U25 is looped out. This creates a major distortion of the phosphate backbone between C24 and G26. In contrast, in the bound TAR RNA, each of the three residues from the bulge are looped out of the helix and U23 is drawn into proximity with G26 through contacts with an arginine residue that is inserted between the two bases. Thus, TAR RNA undergoes a transition from a structure with an open and accessible major groove to a much more tightly packed structure that is folded around basic side chains emanating from the Tat protein.

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Selected References

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  1. Aboul-ela F., Karn J., Varani G. The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein. J Mol Biol. 1995 Oct 20;253(2):313–332. doi: 10.1006/jmbi.1995.0555. [DOI] [PubMed] [Google Scholar]
  2. Aboul-ela F., Murchie A. I., Homans S. W., Lilley D. M. Nuclear magnetic resonance study of a deoxyoligonucleotide duplex containing a three base bulge. J Mol Biol. 1993 Jan 5;229(1):173–188. doi: 10.1006/jmbi.1993.1016. [DOI] [PubMed] [Google Scholar]
  3. Allain F. H., Varani G. Structure of the P1 helix from group I self-splicing introns. J Mol Biol. 1995 Jul 14;250(3):333–353. doi: 10.1006/jmbi.1995.0381. [DOI] [PubMed] [Google Scholar]
  4. Avis J. M., Allain F. H., Howe P. W., Varani G., Nagai K., Neuhaus D. Solution structure of the N-terminal RNP domain of U1A protein: the role of C-terminal residues in structure stability and RNA binding. J Mol Biol. 1996 Mar 29;257(2):398–411. doi: 10.1006/jmbi.1996.0171. [DOI] [PubMed] [Google Scholar]
  5. Batey R. T., Battiste J. L., Williamson J. R. Preparation of isotopically enriched RNAs for heteronuclear NMR. Methods Enzymol. 1995;261:300–322. doi: 10.1016/s0076-6879(95)61015-4. [DOI] [PubMed] [Google Scholar]
  6. Bayer P., Kraft M., Ejchart A., Westendorp M., Frank R., Rösch P. Structural studies of HIV-1 Tat protein. J Mol Biol. 1995 Apr 7;247(4):529–535. doi: 10.1006/jmbi.1995.0158. [DOI] [PubMed] [Google Scholar]
  7. Bhattacharyya A., Lilley D. M. The contrasting structures of mismatched DNA sequences containing looped-out bases (bulges) and multiple mismatches (bubbles). Nucleic Acids Res. 1989 Sep 12;17(17):6821–6840. doi: 10.1093/nar/17.17.6821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bhattacharyya A., Murchie A. I., Lilley D. M. RNA bulges and the helical periodicity of double-stranded RNA. Nature. 1990 Feb 1;343(6257):484–487. doi: 10.1038/343484a0. [DOI] [PubMed] [Google Scholar]
  9. Calnan B. J., Biancalana S., Hudson D., Frankel A. D. Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition. Genes Dev. 1991 Feb;5(2):201–210. doi: 10.1101/gad.5.2.201. [DOI] [PubMed] [Google Scholar]
  10. Churcher M. J., Lamont C., Hamy F., Dingwall C., Green S. M., Lowe A. D., Butler J. G., Gait M. J., Karn J. High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region. J Mol Biol. 1993 Mar 5;230(1):90–110. doi: 10.1006/jmbi.1993.1128. [DOI] [PubMed] [Google Scholar]
  11. Churcher M. J., Lowe A. D., Gait M. J., Karn J. The RNA element encoded by the trans-activation-responsive region of human immunodeficiency virus type 1 is functional when displaced downstream of the start of transcription. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2408–2412. doi: 10.1073/pnas.92.6.2408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cordingley M. G., LaFemina R. L., Callahan P. L., Condra J. H., Sardana V. V., Graham D. J., Nguyen T. M., LeGrow K., Gotlib L., Schlabach A. J. Sequence-specific interaction of Tat protein and Tat peptides with the transactivation-responsive sequence element of human immunodeficiency virus type 1 in vitro. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8985–8989. doi: 10.1073/pnas.87.22.8985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Delling U., Reid L. S., Barnett R. W., Ma M. Y., Climie S., Sumner-Smith M., Sonenberg N. Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure. J Virol. 1992 May;66(5):3018–3025. doi: 10.1128/jvi.66.5.3018-3025.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Diamond R. On the multiple simultaneous superposition of molecular structures by rigid body transformations. Protein Sci. 1992 Oct;1(10):1279–1287. doi: 10.1002/pro.5560011006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dingwall C., Ernberg I., Gait M. J., Green S. M., Heaphy S., Karn J., Lowe A. D., Singh M., Skinner M. A. HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure. EMBO J. 1990 Dec;9(12):4145–4153. doi: 10.1002/j.1460-2075.1990.tb07637.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dingwall C., Ernberg I., Gait M. J., Green S. M., Heaphy S., Karn J., Lowe A. D., Singh M., Skinner M. A., Valerio R. Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6925–6929. doi: 10.1073/pnas.86.18.6925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dougherty D. A. Cation-pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. Science. 1996 Jan 12;271(5246):163–168. doi: 10.1126/science.271.5246.163. [DOI] [PubMed] [Google Scholar]
  18. Feinberg M. B., Baltimore D., Frankel A. D. The role of Tat in the human immunodeficiency virus life cycle indicates a primary effect on transcriptional elongation. Proc Natl Acad Sci U S A. 1991 May 1;88(9):4045–4049. doi: 10.1073/pnas.88.9.4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gohlke C., Murchie A. I., Lilley D. M., Clegg R. M. Kinking of DNA and RNA helices by bulged nucleotides observed by fluorescence resonance energy transfer. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11660–11664. doi: 10.1073/pnas.91.24.11660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Graeble M. A., Churcher M. J., Lowe A. D., Gait M. J., Karn J. Human immunodeficiency virus type 1 transactivator protein, tat, stimulates transcriptional read-through of distal terminator sequences in vitro. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6184–6188. doi: 10.1073/pnas.90.13.6184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Graham G. J., Maio J. J. RNA transcripts of the human immunodeficiency virus transactivation response element can inhibit action of the viral transactivator. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5817–5821. doi: 10.1073/pnas.87.15.5817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hamy F., Asseline U., Grasby J., Iwai S., Pritchard C., Slim G., Butler P. J., Karn J., Gait M. J. Hydrogen-bonding contacts in the major groove are required for human immunodeficiency virus type-1 tat protein recognition of TAR RNA. J Mol Biol. 1993 Mar 5;230(1):111–123. doi: 10.1006/jmbi.1993.1129. [DOI] [PubMed] [Google Scholar]
  23. Harada K., Martin S. S., Frankel A. D. Selection of RNA-binding peptides in vivo. Nature. 1996 Mar 14;380(6570):175–179. doi: 10.1038/380175a0. [DOI] [PubMed] [Google Scholar]
  24. Harrich D., Mavankal G., Mette-Snider A., Gaynor R. B. Human immunodeficiency virus type 1 TAR element revertant viruses define RNA structures required for efficient viral gene expression and replication. J Virol. 1995 Aug;69(8):4906–4913. doi: 10.1128/jvi.69.8.4906-4913.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hsieh C. H., Griffith J. D. Deletions of bases in one strand of duplex DNA, in contrast to single-base mismatches, produce highly kinked molecules: possible relevance to the folding of single-stranded nucleic acids. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4833–4837. doi: 10.1073/pnas.86.13.4833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jucker F. M., Pardi A. Solution structure of the CUUG hairpin loop: a novel RNA tetraloop motif. Biochemistry. 1995 Nov 7;34(44):14416–14427. doi: 10.1021/bi00044a019. [DOI] [PubMed] [Google Scholar]
  27. Kao S. Y., Calman A. F., Luciw P. A., Peterlin B. M. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature. 1987 Dec 3;330(6147):489–493. doi: 10.1038/330489a0. [DOI] [PubMed] [Google Scholar]
  28. Keen N. J., Gait M. J., Karn J. Human immunodeficiency virus type-1 Tat is an integral component of the activated transcription-elongation complex. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2505–2510. doi: 10.1073/pnas.93.6.2505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. King G. C., Harper J. W., Xi Z. Isotope labeling for 13C relaxation measurements on RNA. Methods Enzymol. 1995;261:436–450. doi: 10.1016/s0076-6879(95)61020-0. [DOI] [PubMed] [Google Scholar]
  30. Klaver B., Berkhout B. Evolution of a disrupted TAR RNA hairpin structure in the HIV-1 virus. EMBO J. 1994 Jun 1;13(11):2650–2659. doi: 10.1002/j.1460-2075.1994.tb06555.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Laspia M. F., Rice A. P., Mathews M. B. HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation. Cell. 1989 Oct 20;59(2):283–292. doi: 10.1016/0092-8674(89)90290-0. [DOI] [PubMed] [Google Scholar]
  32. Lazinski D., Grzadzielska E., Das A. Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif. Cell. 1989 Oct 6;59(1):207–218. doi: 10.1016/0092-8674(89)90882-9. [DOI] [PubMed] [Google Scholar]
  33. Lilley D. M. Kinking of DNA and RNA by base bulges. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7140–7142. doi: 10.1073/pnas.92.16.7140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Marciniak R. A., Calnan B. J., Frankel A. D., Sharp P. A. HIV-1 Tat protein trans-activates transcription in vitro. Cell. 1990 Nov 16;63(4):791–802. doi: 10.1016/0092-8674(90)90145-5. [DOI] [PubMed] [Google Scholar]
  35. Marciniak R. A., Sharp P. A. HIV-1 Tat protein promotes formation of more-processive elongation complexes. EMBO J. 1991 Dec;10(13):4189–4196. doi: 10.1002/j.1460-2075.1991.tb04997.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Muesing M. A., Smith D. H., Capon D. J. Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell. 1987 Feb 27;48(4):691–701. doi: 10.1016/0092-8674(87)90247-9. [DOI] [PubMed] [Google Scholar]
  37. Mujeeb A., Parslow T. G., Yuan Y. C., James T. L. Aqueous solution structure of a hybrid lentiviral Tat peptide and a model of its interaction with HIV-1 TAR RNA. J Biomol Struct Dyn. 1996 Feb;13(4):649–660. doi: 10.1080/07391102.1996.10508877. [DOI] [PubMed] [Google Scholar]
  38. Nikonowicz E. P., Sirr A., Legault P., Jucker F. M., Baer L. M., Pardi A. Preparation of 13C and 15N labelled RNAs for heteronuclear multi-dimensional NMR studies. Nucleic Acids Res. 1992 Sep 11;20(17):4507–4513. doi: 10.1093/nar/20.17.4507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pritchard C. E., Grasby J. A., Hamy F., Zacharek A. M., Singh M., Karn J., Gait M. J. Methylphosphonate mapping of phosphate contacts critical for RNA recognition by the human immunodeficiency virus tat and rev proteins. Nucleic Acids Res. 1994 Jul 11;22(13):2592–2600. doi: 10.1093/nar/22.13.2592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Puglisi J. D., Chen L., Frankel A. D., Williamson J. R. Role of RNA structure in arginine recognition of TAR RNA. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3680–3684. doi: 10.1073/pnas.90.8.3680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Puglisi J. D., Tan R., Calnan B. J., Frankel A. D., Williamson J. R. Conformation of the TAR RNA-arginine complex by NMR spectroscopy. Science. 1992 Jul 3;257(5066):76–80. doi: 10.1126/science.1621097. [DOI] [PubMed] [Google Scholar]
  42. Riordan F. A., Bhattacharyya A., McAteer S., Lilley D. M. Kinking of RNA helices by bulged bases, and the structure of the human immunodeficiency virus transactivator response element. J Mol Biol. 1992 Jul 20;226(2):305–310. doi: 10.1016/0022-2836(92)90947-i. [DOI] [PubMed] [Google Scholar]
  43. Rittner K., Churcher M. J., Gait M. J., Karn J. The human immunodeficiency virus long terminal repeat includes a specialised initiator element which is required for Tat-responsive transcription. J Mol Biol. 1995 May 5;248(3):562–580. doi: 10.1006/jmbi.1995.0243. [DOI] [PubMed] [Google Scholar]
  44. Roy S., Delling U., Chen C. H., Rosen C. A., Sonenberg N. A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation. Genes Dev. 1990 Aug;4(8):1365–1373. doi: 10.1101/gad.4.8.1365. [DOI] [PubMed] [Google Scholar]
  45. Roy S., Parkin N. T., Rosen C., Itovitch J., Sonenberg N. Structural requirements for trans activation of human immunodeficiency virus type 1 long terminal repeat-directed gene expression by tat: importance of base pairing, loop sequence, and bulges in the tat-responsive sequence. J Virol. 1990 Mar;64(3):1402–1406. doi: 10.1128/jvi.64.3.1402-1406.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sullenger B. A., Gallardo H. F., Ungers G. E., Gilboa E. Analysis of trans-acting response decoy RNA-mediated inhibition of human immunodeficiency virus type 1 transactivation. J Virol. 1991 Dec;65(12):6811–6816. doi: 10.1128/jvi.65.12.6811-6816.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sullenger B. A., Gallardo H. F., Ungers G. E., Gilboa E. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell. 1990 Nov 2;63(3):601–608. doi: 10.1016/0092-8674(90)90455-n. [DOI] [PubMed] [Google Scholar]
  48. Sumner-Smith M., Roy S., Barnett R., Reid L. S., Kuperman R., Delling U., Sonenberg N. Critical chemical features in trans-acting-responsive RNA are required for interaction with human immunodeficiency virus type 1 Tat protein. J Virol. 1991 Oct;65(10):5196–5202. doi: 10.1128/jvi.65.10.5196-5202.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tan R., Frankel A. D. Structural variety of arginine-rich RNA-binding peptides. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5282–5286. doi: 10.1073/pnas.92.12.5282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tao J., Frankel A. D. Electrostatic interactions modulate the RNA-binding and transactivation specificities of the human immunodeficiency virus and simian immunodeficiency virus Tat proteins. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1571–1575. doi: 10.1073/pnas.90.4.1571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tao J., Frankel A. D. Specific binding of arginine to TAR RNA. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2723–2726. doi: 10.1073/pnas.89.7.2723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Varani G., Aboul-ela F., Allain F., Gubser C. C. Novel three-dimensional 1H-13C-31P triple resonance experiments for sequential backbone correlations in nucleic acids. J Biomol NMR. 1995 Apr;5(3):315–320. doi: 10.1007/BF00211759. [DOI] [PubMed] [Google Scholar]
  53. Varani G., Tinoco I., Jr RNA structure and NMR spectroscopy. Q Rev Biophys. 1991 Nov;24(4):479–532. doi: 10.1017/s0033583500003875. [DOI] [PubMed] [Google Scholar]
  54. Weeks K. M., Ampe C., Schultz S. C., Steitz T. A., Crothers D. M. Fragments of the HIV-1 Tat protein specifically bind TAR RNA. Science. 1990 Sep 14;249(4974):1281–1285. doi: 10.1126/science.2205002. [DOI] [PubMed] [Google Scholar]
  55. Weeks K. M., Crothers D. M. Major groove accessibility of RNA. Science. 1993 Sep 17;261(5128):1574–1577. doi: 10.1126/science.7690496. [DOI] [PubMed] [Google Scholar]
  56. Weeks K. M., Crothers D. M. RNA binding assays for Tat-derived peptides: implications for specificity. Biochemistry. 1992 Oct 27;31(42):10281–10287. doi: 10.1021/bi00157a015. [DOI] [PubMed] [Google Scholar]
  57. Weeks K. M., Crothers D. M. RNA recognition by Tat-derived peptides: interaction in the major groove? Cell. 1991 Aug 9;66(3):577–588. doi: 10.1016/0092-8674(81)90020-9. [DOI] [PubMed] [Google Scholar]
  58. Weisenseel J. P., Moe J. G., Reddy G. R., Marnett L. J., Stone M. P. Structure of a duplex oligodeoxynucleotide containing propanodeoxyguanosine opposite a two-base deletion in the (CpG)3 frame shift hotspot of Salmonella typhimurium hisD3052 determined by 1H NMR and restrained molecular dynamics. Biochemistry. 1995 Jan 10;34(1):50–64. doi: 10.1021/bi00001a007. [DOI] [PubMed] [Google Scholar]
  59. 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]
  60. Zacharias M., Hagerman P. J. Bulge-induced bends in RNA: quantification by transient electric birefringence. J Mol Biol. 1995 Mar 31;247(3):486–500. doi: 10.1006/jmbi.1995.0155. [DOI] [PubMed] [Google Scholar]
  61. Zacharias M., Hagerman P. J. The bend in RNA created by the trans-activation response element bulge of human immunodeficiency virus is straightened by arginine and by Tat-derived peptide. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6052–6056. doi: 10.1073/pnas.92.13.6052. [DOI] [PMC free article] [PubMed] [Google Scholar]

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