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A simple and rapid TAR-dependent in vitro transcription assay using T cell nuclear extracts and synthetictat1-86 protein

  • Original Paper
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Journal of Biomedical Science

Abstract

A ‘G-less’ cassette was cloned downstream of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) to facilitate rapid identification of authentic LTR-directed transcription in T cell nuclear extracts. Despite a high constitutive level of transcription, addition of chemically synthesized full-length HIV-1bru tat (amino acids 1–86) stimulated transcription 3-fold but only if the template included the TAR region of the LTR. Suppression of basal transcription in T cell nuclear extracts by sodium citrate increased the relative level oftat stimulation, but neither potassium chloride nor histone H1 had an effect. A mutant synthetictat polypeptide, lacking residues 22–32 in the cysteine-rich domain, was inactive in uptake assays and failed to stimulate transcription. Short basic peptides that competed full-lengthtat from complexes with TAR RNA also inhibitedtat stimulation of transcription, whereas short basic peptide unable to bind TAR or competetat from complexes were also unable to inhibittat stimulation of transcription. These data confirm that active HIV-1tat must first interact with TAR RNA via basic amino acid residues in order to stimulate transcription of downstream sequences.

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Authors and Affiliations

  1. Wellcome Research Laboratories, Gene Targets Group, BR3 3BS, Beckenham, Kent, UK

    Edward D. Blair & Christopher M. Roberts

  2. The Biomedical Research Centre, Department of Biochemistry, University of British Columbia, Vancouver, Canada

    Ian Clark-Lewis

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Blair, E.D., Roberts, C.M. & Clark-Lewis, I. A simple and rapid TAR-dependent in vitro transcription assay using T cell nuclear extracts and synthetictat1-86 protein. J Biomed Sci 2, 136–145 (1995). https://doi.org/10.1007/BF02253064

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