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A novel RNA polymerase II-containing complex potentiates Tat-enhanced HIV-1 transcription in vitro.
Parada CA; Roeder RG; Laboratory of Biochemistry and Molecular Biology,
December 30, 1998
HIV Pathog Treat Conf. 1998 Mar 13-19;:43 (abstract no. 1017). Unique

Enhanced HIV-1 gene expression requires virally-encoded Tat, which binds to a specific site (TAR) near the 5' end of the nascent HIV-1 transcript and potentiates transcription elongation. We and others showed previously that Tat function involves increased phosphorylation of the carboxy terminal domain (CTD) of RNA polymerase II (Pol II) by TFIIH, which correlates with enhanced processivity of Pol II. Recent studies by others have also implicated P-TEFb, an elongation factor with CTD kinase activity, in Tat activation and thus Pol II processivity. Here we describe a Tat stimulatory activity (Tat-SF), analogous to that reported by Zhou and Sharp, that is required in addition to TFIIH and P-TEFb for Tat-enhanced HIV-1 transcription in a reconstituted in vitro transcription system. However, we now show that Tat-SF is a novel RNA Pol II complex containing two previously identified Tat-cofactors, Tat-SF1 and PITALRE (a kinase subunit of P-TEFb), as well as other polypeptides. In contrast to the conventional "holo" RNA Pol II complex, Tat-SF contains no SRBs (considered the hallmark of human RNA Pol II holoenzyme), P-CAF, CBP, RHA, or any of the RNA Pol II general transcription factors. Furthermore, we show that these two distinct RNA Pol II-containing complexes co-exist in unfractionated HeLa nuclear extract. Interestingly, substitution of Tat-SF for either "core" or conventional "holo" RNA Pol II is sufficient to support Tat-activated HIV-1 transcription in a purified transcription system, and a Tat-SF-derived complex lacking RNA Pol II can mediate Tat function in conjunction with other RNA Pol II complexes. These results strongly suggest a role for Tat-SF associated polypeptides in RNA Pol II processivity, possibly by triggering CTD phosphorylation by TFIIH and/or P-TEFb. In summary, we demonstrate the coexistence of distinct RNA Pol II complexes in HeLa nuclear extract, and that Tat utilizes only one of these to potentiate HIV-1 transcription elongation.

MEETING ABSTRACTS Gene Products, tat/*METABOLISM Hela Cells Human HIV-1/*GENETICS Phosphorylation RNA Polymerase II/*METABOLISM *Transcription, Genetic

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