Analysis of Tat transactivation of human immunodeficiency virus transcription in vitro.

C. A. Bohan, F. Kashanchi, B. Ensoli, L. Buonaguro, K. A. Boris-Lawrie, J. N. Brady

Research output: Contribution to journalArticle

Abstract

The HIV Tat protein is a potent transactivator of HIV transcription, increasing both RNA initiation and elongation. We now demonstrate that purified, full-length 86 amino acid Tat protein specifically transactivates the HIV LTR in vitro to a high level (25- to 60-fold). Tat transactivation was specifically blocked by anti-Tat serum, but not preimmune serum. Tat did not transactivate transcription from the control adenovirus major late promoter (AdMLP). HIV transcription was blocked at various functional steps during initiation and elongation complex formation. Similar to the control AdMLP, HIV basal initiation complex assembly was sensitive to the addition of 0.015% sarkosyl prior to the addition of nucleoside triphosphates. Resistance to 0.05% sarkosyl required the addition of G, C, and U, which constitute the first 13 bases of the HIV RNA transcript. The addition of Tat to the in vitro transcription relieved the 0.015% sarkosyl block. These Tat-induced complexes were sensitive to 0.05% sarkosyl, suggesting that transcriptional initiation had not occurred. Consistent with this hypothesis, the addition of G, C, and U to the Tat-induced transcription complexes allowed the rapid conversion to transcription initiation complexes. Tat also facilitated the formation of 0.015% sarkosyl-resistant complexes in a reconstituted transcription system containing partially purified transcription factors and polymerase II. Following the formation of stable initiation complexes, Tat increased the rate and efficiency of transcription elongation on the HIV but not the AdML template. Kinetic analysis of Tat transactivation suggests that approximately 30% of the Tat initiation complexes are converted to elongation complexes. We conclude that Tat, in addition to its demonstrated role in RNA elongation, facilitates transcription initiation in vitro.

Original languageEnglish
Pages (from-to)391-407
Number of pages17
JournalGene Expression
Volume2
Issue number4
Publication statusPublished - 1992

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Transcriptional Activation
HIV
RNA
Adenoviridae
Human Immunodeficiency Virus tat Gene Products
tat Gene Products
Trans-Activators
Serum
Nucleosides
In Vitro Techniques
Transcription Factors
sarkosyl
Amino Acids

ASJC Scopus subject areas

  • Genetics

Cite this

Bohan, C. A., Kashanchi, F., Ensoli, B., Buonaguro, L., Boris-Lawrie, K. A., & Brady, J. N. (1992). Analysis of Tat transactivation of human immunodeficiency virus transcription in vitro. Gene Expression, 2(4), 391-407.

Analysis of Tat transactivation of human immunodeficiency virus transcription in vitro. / Bohan, C. A.; Kashanchi, F.; Ensoli, B.; Buonaguro, L.; Boris-Lawrie, K. A.; Brady, J. N.

In: Gene Expression, Vol. 2, No. 4, 1992, p. 391-407.

Research output: Contribution to journalArticle

Bohan, CA, Kashanchi, F, Ensoli, B, Buonaguro, L, Boris-Lawrie, KA & Brady, JN 1992, 'Analysis of Tat transactivation of human immunodeficiency virus transcription in vitro.', Gene Expression, vol. 2, no. 4, pp. 391-407.
Bohan CA, Kashanchi F, Ensoli B, Buonaguro L, Boris-Lawrie KA, Brady JN. Analysis of Tat transactivation of human immunodeficiency virus transcription in vitro. Gene Expression. 1992;2(4):391-407.
Bohan, C. A. ; Kashanchi, F. ; Ensoli, B. ; Buonaguro, L. ; Boris-Lawrie, K. A. ; Brady, J. N. / Analysis of Tat transactivation of human immunodeficiency virus transcription in vitro. In: Gene Expression. 1992 ; Vol. 2, No. 4. pp. 391-407.
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