Specific binding of RNA polymerase II to the human immunodeficiency virus trans-activating region RNA is regulated by cellular cofactors and Tat.
The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression in response to Tat is dependent on an element downstream of the HIV-1 transcriptional initiation site designated the trans-activating region (TAR). TAR forms a stable stem-loop RNA structure in which a 3-nt bulge structure and a 6-nt loop structure are important for Tat activation. In the absence of Tat, the HIV-1 promoter generates so-called short or nonprocessive transcripts terminating at +60, while in the presence of Tat the synthesis of these short transcripts is markedly decreased and transcripts that extend through the 9.0-kb HIV-1 genome are synthesized. Tat effects on transcriptional elongation are likely due to alterations in the elongation properties of RNA polymerase II. In this study we demonstrated that a set of cellular cofactors that modulate the binding of the cellular protein TRP-185 to the TAR RNA loop sequences also functioned to markedly stimulate the specific binding of hypophosphorylated (IIa) and hyperphosphorylated (IIo) RNA polymerase II to TAR RNA. The concentrations of RNA polymerase II required for this interaction with TAR RNA were similar to those required to initiate in vitro transcription from the HIV-1 long terminal repeat. RNA gel retardation analysis with wild-type and mutant TAR RNAs indicated that the TAR RNA loop and bulge sequences were critical for the binding of RNA polymerase II. The addition of wild-type but not mutant Tat protein to gel retardation analysis with TAR RNA and RNA polymerase II resulted in the loss of binding of RNA polymerase II binding to TAR RNA. These results suggest that Tat may function to alter RNA polymerase II, which is paused due to its binding to HIV-1 TAR RNA with resultant stimulation of its transcriptional elongation properties.
Wu-Baer
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F
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8594, USA.
Sigman
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D
Gaynor
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RB
D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000378:metabolism*; D015483:HIV Antibodies; D015497:HIV-1 / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000276:immunology / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D015483:HIV Antibodies; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; D012319:RNA Polymerase II