Transcriptional trans activation by human immunodeficiency virus type 1 Tat requires specific coactivators that are not basal factors.
Expression of human immunodeficiency virus type 1 (HIV-1) genes is regulated by the trans activator Tat. Tat exerts its effects by increasing the rate of transcription, but the mechanism by which it does so is still unknown. To study the cellular factors required for Tat trans activation, we have expressed functional Gst-Tat fusion protein and used it to construct affinity columns. Our findings are as follows. (i) A Gst-Tat affinity matrix depleted HeLa nuclear extracts of a factor(s) required for Tat function. A Tat mutant bearing the missense mutation lysine to alanine at position 41 was incapable of this depletion. (ii) Tat trans activation was recovered by addition of unfractionated nuclear extract, the 0.5 M KCl elution fraction from the Tat affinity column, or sedimentation gradient fractions of HeLa extracts. The activity from the gradients sedimented with an apparent molecular mass of 200 kDa. (iii) Tat trans activation could not be recovered by use of recombinant human TATA-binding protein or partially purified TFIID. (iv) trans activation by Tat was blocked by heating of the nuclear extract under conditions in which basal transcription was not decreased. Our data demonstrate for the first time the existence of unique Tat coactivators distinct from factors required for general basal transcription.
Suñé
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Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
García-Blanco
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D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000235:genetics* / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D035181:TATA-Box Binding Protein; D013696:Temperature; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D004268:DNA-Binding Proteins; D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; D005982:Glutathione Transferase