Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Dec;17(12):6898–6905. doi: 10.1128/mcb.17.12.6898

Promoter activity of Tat at steps subsequent to TATA-binding protein recruitment.

H Xiao 1, J T Lis 1, K T Jeang 1
PMCID: PMC232546  PMID: 9372921

Abstract

Artificial recruitment of TATA-binding protein (TBP) to many eukaryotic promoters bypasses DNA-bound activator function. The human immunodeficiency virus type 1 (HIV-1) Tat is an unconventional activator that up-regulates transcription from the HIV-1 long terminal repeat (LTR) through binding to a nascent RNA sequence, TAR. Because this LTR and its cognate activator have atypical features compared to a standard RNA polymerase II (RNAP II) transcriptional unit, the precise limiting steps for HIV-1 transcription and how Tat resolves these limitations remain incompletely understood. We thus constructed human TBP fused to the DNA-binding domain of GAL4 to determine whether recruitment of TBP is one rate-limiting step in HIV-1 LTR transcription and whether Tat functions to recruit TBP. As a control, we compared the activity of the adenovirus E1b promoter. Our findings indicate that TBP tethering to the E1b promoter fully effected transcription to the same degree achievable with the potent GAL4-VP16 activator. By contrast, TBP recruitment to the HIV-1 LTR, although necessary for conferring Tat responsiveness, did not bypass a physical need for Tat in achieving activated transcription. These results document that the HIV-1 and the E1b promoters are transcriptionally limited at different steps; the major rate-limiting step for E1b is recruitment of TBP, while activation of the HIV-1 LTR requires steps in addition to TBP recruitment. We suggest that Tat acts to accelerate rate-limiting steps after TBP recruitment.

Full Text

The Full Text of this article is available as a PDF (903.4 KB).

Selected References

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

  1. Barberis A., Pearlberg J., Simkovich N., Farrell S., Reinagel P., Bamdad C., Sigal G., Ptashne M. Contact with a component of the polymerase II holoenzyme suffices for gene activation. Cell. 1995 May 5;81(3):359–368. doi: 10.1016/0092-8674(95)90389-5. [DOI] [PubMed] [Google Scholar]
  2. Berkhout B., Gatignol A., Rabson A. B., Jeang K. T. TAR-independent activation of the HIV-1 LTR: evidence that tat requires specific regions of the promoter. Cell. 1990 Aug 24;62(4):757–767. doi: 10.1016/0092-8674(90)90120-4. [DOI] [PubMed] [Google Scholar]
  3. Berkhout B., Jeang K. T. Detailed mutational analysis of TAR RNA: critical spacing between the bulge and loop recognition domains. Nucleic Acids Res. 1991 Nov 25;19(22):6169–6176. doi: 10.1093/nar/19.22.6169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berkhout B., Jeang K. T. Functional roles for the TATA promoter and enhancers in basal and Tat-induced expression of the human immunodeficiency virus type 1 long terminal repeat. J Virol. 1992 Jan;66(1):139–149. doi: 10.1128/jvi.66.1.139-149.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berkhout B., Silverman R. H., Jeang K. T. Tat trans-activates the human immunodeficiency virus through a nascent RNA target. Cell. 1989 Oct 20;59(2):273–282. doi: 10.1016/0092-8674(89)90289-4. [DOI] [PubMed] [Google Scholar]
  6. Blau J., Xiao H., McCracken S., O'Hare P., Greenblatt J., Bentley D. Three functional classes of transcriptional activation domain. Mol Cell Biol. 1996 May;16(5):2044–2055. doi: 10.1128/mcb.16.5.2044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buratowski S., Hahn S., Guarente L., Sharp P. A. Five intermediate complexes in transcription initiation by RNA polymerase II. Cell. 1989 Feb 24;56(4):549–561. doi: 10.1016/0092-8674(89)90578-3. [DOI] [PubMed] [Google Scholar]
  8. Burley S. K., Roeder R. G. Biochemistry and structural biology of transcription factor IID (TFIID). Annu Rev Biochem. 1996;65:769–799. doi: 10.1146/annurev.bi.65.070196.004005. [DOI] [PubMed] [Google Scholar]
  9. Chao D. M., Gadbois E. L., Murray P. J., Anderson S. F., Sonu M. S., Parvin J. D., Young R. A. A mammalian SRB protein associated with an RNA polymerase II holoenzyme. Nature. 1996 Mar 7;380(6569):82–85. doi: 10.1038/380082a0. [DOI] [PubMed] [Google Scholar]
  10. Chatterjee S., Struhl K. Connecting a promoter-bound protein to TBP bypasses the need for a transcriptional activation domain. Nature. 1995 Apr 27;374(6525):820–822. doi: 10.1038/374820a0. [DOI] [PubMed] [Google Scholar]
  11. Chun R. F., Jeang K. T. Requirements for RNA polymerase II carboxyl-terminal domain for activated transcription of human retroviruses human T-cell lymphotropic virus I and HIV-1. J Biol Chem. 1996 Nov 1;271(44):27888–27894. doi: 10.1074/jbc.271.44.27888. [DOI] [PubMed] [Google Scholar]
  12. Cujec T. P., Cho H., Maldonado E., Meyer J., Reinberg D., Peterlin B. M. The human immunodeficiency virus transactivator Tat interacts with the RNA polymerase II holoenzyme. Mol Cell Biol. 1997 Apr;17(4):1817–1823. doi: 10.1128/mcb.17.4.1817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Emili A., Greenblatt J., Ingles C. J. Species-specific interaction of the glutamine-rich activation domains of Sp1 with the TATA box-binding protein. Mol Cell Biol. 1994 Mar;14(3):1582–1593. doi: 10.1128/mcb.14.3.1582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. García-Martínez L. F., Ivanov D., Gaynor R. B. Association of Tat with purified HIV-1 and HIV-2 transcription preinitiation complexes. J Biol Chem. 1997 Mar 14;272(11):6951–6958. doi: 10.1074/jbc.272.11.6951. [DOI] [PubMed] [Google Scholar]
  16. Gaudreau L., Schmid A., Blaschke D., Ptashne M., Hörz W. RNA polymerase II holoenzyme recruitment is sufficient to remodel chromatin at the yeast PHO5 promoter. Cell. 1997 Apr 4;89(1):55–62. doi: 10.1016/s0092-8674(00)80182-8. [DOI] [PubMed] [Google Scholar]
  17. Ghosh S., Selby M. J., Peterlin B. M. Synergism between Tat and VP16 in trans-activation of HIV-1 LTR. J Mol Biol. 1993 Dec 5;234(3):610–619. doi: 10.1006/jmbi.1993.1615. [DOI] [PubMed] [Google Scholar]
  18. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Green S., Issemann I., Sheer E. A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Res. 1988 Jan 11;16(1):369–369. doi: 10.1093/nar/16.1.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Herrmann C. H., Gold M. O., Rice A. P. Viral transactivators specifically target distinct cellular protein kinases that phosphorylate the RNA polymerase II C-terminal domain. Nucleic Acids Res. 1996 Feb 1;24(3):501–508. doi: 10.1093/nar/24.3.501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Huang L. M., Jeang K. T. Increased spacing between Sp1 and TATAA renders human immunodeficiency virus type 1 replication defective: implication for Tat function. J Virol. 1993 Dec;67(12):6937–6944. doi: 10.1128/jvi.67.12.6937-6944.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ingles C. J., Shales M., Cress W. D., Triezenberg S. J., Greenblatt J. Reduced binding of TFIID to transcriptionally compromised mutants of VP16. Nature. 1991 Jun 13;351(6327):588–590. doi: 10.1038/351588a0. [DOI] [PubMed] [Google Scholar]
  23. Jeang K. T., Berkhout B., Dropulic B. Effects of integration and replication on transcription of the HIV-1 long terminal repeat. J Biol Chem. 1993 Nov 25;268(33):24940–24949. [PubMed] [Google Scholar]
  24. Jeang K. T., Chun R., Lin N. H., Gatignol A., Glabe C. G., Fan H. In vitro and in vivo binding of human immunodeficiency virus type 1 Tat protein and Sp1 transcription factor. J Virol. 1993 Oct;67(10):6224–6233. doi: 10.1128/jvi.67.10.6224-6233.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Joliot V., Demma M., Prywes R. Interaction with RAP74 subunit of TFIIF is required for transcriptional activation by serum response factor. Nature. 1995 Feb 16;373(6515):632–635. doi: 10.1038/373632a0. [DOI] [PubMed] [Google Scholar]
  26. Klages N., Strubin M. Stimulation of RNA polymerase II transcription initiation by recruitment of TBP in vivo. Nature. 1995 Apr 27;374(6525):822–823. doi: 10.1038/374822a0. [DOI] [PubMed] [Google Scholar]
  27. Kobayashi N., Boyer T. G., Berk A. J. A class of activation domains interacts directly with TFIIA and stimulates TFIIA-TFIID-promoter complex assembly. Mol Cell Biol. 1995 Nov;15(11):6465–6473. doi: 10.1128/mcb.15.11.6465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Koleske A. J., Young R. A. An RNA polymerase II holoenzyme responsive to activators. Nature. 1994 Mar 31;368(6470):466–469. doi: 10.1038/368466a0. [DOI] [PubMed] [Google Scholar]
  29. Krumm A., Meulia T., Brunvand M., Groudine M. The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region. Genes Dev. 1992 Nov;6(11):2201–2213. doi: 10.1101/gad.6.11.2201. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Lillie J. W., Green M. R. Transcription activation by the adenovirus E1a protein. Nature. 1989 Mar 2;338(6210):39–44. doi: 10.1038/338039a0. [DOI] [PubMed] [Google Scholar]
  32. Lin Y. S., Ha I., Maldonado E., Reinberg D., Green M. R. Binding of general transcription factor TFIIB to an acidic activating region. Nature. 1991 Oct 10;353(6344):569–571. doi: 10.1038/353569a0. [DOI] [PubMed] [Google Scholar]
  33. Lu X., Welsh T. M., Peterlin B. M. The human immunodeficiency virus type 1 long terminal repeat specifies two different transcription complexes, only one of which is regulated by Tat. J Virol. 1993 Apr;67(4):1752–1760. doi: 10.1128/jvi.67.4.1752-1760.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Mavankal G., Ignatius Ou S. H., Oliver H., Sigman D., Gaynor R. B. Human immunodeficiency virus type 1 and 2 Tat proteins specifically interact with RNA polymerase II. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):2089–2094. doi: 10.1073/pnas.93.5.2089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  37. Okamoto H., Sheline C. T., Corden J. L., Jones K. A., Peterlin B. M. Trans-activation by human immunodeficiency virus Tat protein requires the C-terminal domain of RNA polymerase II. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11575–11579. doi: 10.1073/pnas.93.21.11575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ossipow V., Tassan J. P., Nigg E. A., Schibler U. A mammalian RNA polymerase II holoenzyme containing all components required for promoter-specific transcription initiation. Cell. 1995 Oct 6;83(1):137–146. doi: 10.1016/0092-8674(95)90242-2. [DOI] [PubMed] [Google Scholar]
  39. Parada C. A., Roeder R. G. Enhanced processivity of RNA polymerase II triggered by Tat-induced phosphorylation of its carboxy-terminal domain. Nature. 1996 Nov 28;384(6607):375–378. doi: 10.1038/384375a0. [DOI] [PubMed] [Google Scholar]
  40. Pendergrast P. S., Hernandez N. RNA-targeted activators, but not DNA-targeted activators, repress the synthesis of short transcripts at the human immunodeficiency virus type 1 long terminal repeat. J Virol. 1997 Feb;71(2):910–917. doi: 10.1128/jvi.71.2.910-917.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ptashne M., Gann A. Transcriptional activation by recruitment. Nature. 1997 Apr 10;386(6625):569–577. doi: 10.1038/386569a0. [DOI] [PubMed] [Google Scholar]
  42. Rougvie A. E., Lis J. T. The RNA polymerase II molecule at the 5' end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged. Cell. 1988 Sep 9;54(6):795–804. doi: 10.1016/s0092-8674(88)91087-2. [DOI] [PubMed] [Google Scholar]
  43. Seipel K., Georgiev O., Gerber H. P., Schaffner W. C-terminal domain (CTD) of RNA-polymerase II and N-terminal segment of the human TATA binding protein (TBP) can mediate remote and proximal transcriptional activation, respectively. Nucleic Acids Res. 1993 Dec 11;21(24):5609–5615. doi: 10.1093/nar/21.24.5609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Seipel K., Georgiev O., Schaffner W. Different activation domains stimulate transcription from remote ('enhancer') and proximal ('promoter') positions. EMBO J. 1992 Dec;11(13):4961–4968. doi: 10.1002/j.1460-2075.1992.tb05603.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Selby M. J., Bain E. S., Luciw P. A., Peterlin B. M. Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat. Genes Dev. 1989 Apr;3(4):547–558. doi: 10.1101/gad.3.4.547. [DOI] [PubMed] [Google Scholar]
  46. Stargell L. A., Struhl K. A new class of activation-defective TATA-binding protein mutants: evidence for two steps of transcriptional activation in vivo. Mol Cell Biol. 1996 Aug;16(8):4456–4464. doi: 10.1128/mcb.16.8.4456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Stargell L. A., Struhl K. Mechanisms of transcriptional activation in vivo: two steps forward. Trends Genet. 1996 Aug;12(8):311–315. doi: 10.1016/0168-9525(96)10028-7. [DOI] [PubMed] [Google Scholar]
  48. Stringer K. F., Ingles C. J., Greenblatt J. Direct and selective binding of an acidic transcriptional activation domain to the TATA-box factor TFIID. Nature. 1990 Jun 28;345(6278):783–786. doi: 10.1038/345783a0. [DOI] [PubMed] [Google Scholar]
  49. Strubin M., Struhl K. Yeast and human TFIID with altered DNA-binding specificity for TATA elements. Cell. 1992 Feb 21;68(4):721–730. doi: 10.1016/0092-8674(92)90147-5. [DOI] [PubMed] [Google Scholar]
  50. Struhl K. Chromatin structure and RNA polymerase II connection: implications for transcription. Cell. 1996 Jan 26;84(2):179–182. doi: 10.1016/s0092-8674(00)80970-8. [DOI] [PubMed] [Google Scholar]
  51. Tansey W. P., Ruppert S., Tjian R., Herr W. Multiple regions of TBP participate in the response to transcriptional activators in vivo. Genes Dev. 1994 Nov 15;8(22):2756–2769. doi: 10.1101/gad.8.22.2756. [DOI] [PubMed] [Google Scholar]
  52. Tjian R., Maniatis T. Transcriptional activation: a complex puzzle with few easy pieces. Cell. 1994 Apr 8;77(1):5–8. doi: 10.1016/0092-8674(94)90227-5. [DOI] [PubMed] [Google Scholar]
  53. Workman J. L., Taylor I. C., Kingston R. E. Activation domains of stably bound GAL4 derivatives alleviate repression of promoters by nucleosomes. Cell. 1991 Feb 8;64(3):533–544. doi: 10.1016/0092-8674(91)90237-s. [DOI] [PubMed] [Google Scholar]
  54. Xiao H., Friesen J. D., Lis J. T. Recruiting TATA-binding protein to a promoter: transcriptional activation without an upstream activator. Mol Cell Biol. 1995 Oct;15(10):5757–5761. doi: 10.1128/mcb.15.10.5757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Xiao H., Pearson A., Coulombe B., Truant R., Zhang S., Regier J. L., Triezenberg S. J., Reinberg D., Flores O., Ingles C. J. Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol Cell Biol. 1994 Oct;14(10):7013–7024. doi: 10.1128/mcb.14.10.7013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yankulov K., Blau J., Purton T., Roberts S., Bentley D. L. Transcriptional elongation by RNA polymerase II is stimulated by transactivators. Cell. 1994 Jun 3;77(5):749–759. doi: 10.1016/0092-8674(94)90058-2. [DOI] [PubMed] [Google Scholar]
  57. Zawel L., Kumar K. P., Reinberg D. Recycling of the general transcription factors during RNA polymerase II transcription. Genes Dev. 1995 Jun 15;9(12):1479–1490. doi: 10.1101/gad.9.12.1479. [DOI] [PubMed] [Google Scholar]
  58. Zawel L., Reinberg D. Common themes in assembly and function of eukaryotic transcription complexes. Annu Rev Biochem. 1995;64:533–561. doi: 10.1146/annurev.bi.64.070195.002533. [DOI] [PubMed] [Google Scholar]
  59. Zhou Q., Sharp P. A. Novel mechanism and factor for regulation by HIV-1 Tat. EMBO J. 1995 Jan 16;14(2):321–328. doi: 10.1002/j.1460-2075.1995.tb07006.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zhou Q., Sharp P. A. Tat-SF1: cofactor for stimulation of transcriptional elongation by HIV-1 Tat. Science. 1996 Oct 25;274(5287):605–610. doi: 10.1126/science.274.5287.605. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES