HIV-1 Tat Overcomes Inefficient Transcriptional Elongation in Vitro

Laspia, Michael F.; Wendel, Patricia; Mathews, Michael B.

doi:10.1006/jmbi.1993.1427

Cited by 60 publications

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“…This suggested that Tat phosphorylation might influence its ability to trans-activate via its interaction with TAR, or via effects on its cellular localization, or both. When tested in a Tat-dependent transcription assay (65), phosphorylation by PKR elicited no discernible effect on trans-activation 2 ; however, this negative result is difficult to interpret as only a fraction of the Tat was modified. On the other hand, in this study we demonstrate the phosphorylation of Tat by the mitogen-activated kinase PKC.…”

Section: Discussionmentioning

confidence: 99%

The Tat Protein of Human Immunodeficiency Virus Type 1 Is a Substrate and Inhibitor of the Interferon-induced, Virally Activated Protein Kinase, PKR

Brand

Kobayashi

Mathews

1997

Journal of Biological Chemistry

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135

102

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Section: Discussionmentioning

confidence: 99%

The Tat Protein of Human Immunodeficiency Virus Type 1 Is a Substrate and Inhibitor of the Interferon-induced, Virally Activated Protein Kinase, PKR

Brand

Kobayashi

Mathews

1997

Journal of Biological Chemistry

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135

102

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“…Upon activation by heat shock, Pol II is able to rapidly transcribe these genes. Regulation of Pol II activity after recruitment has also been described in bacteria 23 , yeast 13 and mammalian cell lines 3,[6][7][8][9][10][11][12] , and includes instances where Pol II is found in an inactive pre-initiation complex 24,25 . We will collectively refer to inactive Pol II near the transcription start site as stalled Pol II.…”

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confidence: 95%

RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo

et al. 2007

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It is widely assumed that the key rate-limiting step in gene activation is the recruitment of RNA polymerase II (Pol II) to the core promoter 2 . Although there are well-documented examples where Pol II is recruited to a gene but stalls3 -14, a general role for Pol II stalling in development has not been established. We have performed comprehensive Pol II ChIP-chip assays in Drosophila embryos and identified three distinct Pol II binding behaviors: active (uniform binding across the entire transcription unit), no binding, and stalled (binding at the transcription start site). The striking feature of the ~10% genes that are stalled is that they are highly enriched for developmental control genes that are repressed at the time of analysis or poised for activation during subsequent stages of development. We propose that Pol II stalling facilitates rapid temporal and spatial changes in gene activity during development.Pol II stalling is probably best studied at heat shock genes in Drosophila, where Pol II engages in transcription but pauses immediately downstream of the transcriptional start site 3,4,22 . Upon activation by heat shock, Pol II is able to rapidly transcribe these genes. Regulation of Pol II activity after recruitment has also been described in bacteria 23 , yeast 13 and mammalian cell lines 3,[6][7][8][9][10][11][12] , and includes instances where Pol II is found in an inactive pre-initiation complex 24,25 . We will collectively refer to inactive Pol II near the transcription start site as stalled Pol II.To determine at which genes Pol II stalling occurs during development, we analyzed global Pol II occupancy in whole Drosophila embryos. While this is one of the few systems where The results show that many genes known to be repressed in Toll 10b embryos display strikingly high levels of Pol II near the transcription start site (Fig.1A-D). In some cases the prominent Pol II peak is tightly restricted to the promoter region (e.g. at the tup gene in Fig. 1A), while at other genes Pol II is also found at low levels throughout the transcription unit (e.g. the sog and brk genes Fig. 1C,D). This is consistent with previous evidence that some genes such as sog are transiently activated but then repressed at later stages 26 , while other genes such as tup are never activated in Toll 10b mutants 19,20 .The Pol II profile of repressed genes is clearly distinct from those of active genes ( Fig. 1E, F). For example, the Hbr gene, which encodes an FGF receptor specifically expressed in mesodermal precursors ( Fig. 1E), and ribosomal genes such as RpL3 (Fig.1F), show uniformly high levels of Pol II throughout the transcription unit. Furthermore, genes that are silent in the early embryo simply lack Pol II binding altogether ( Fig. 1G, H). Thus, there appears to be three distinct classes of genes: those with Pol II distributed throughout the transcription unit, those genes with preferential enrichment of Pol II at the transcription site, and genes that lack Pol II binding altogether.To further characterize t...

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“…Tat is able to stimulate elongation, in some cases without any apparent effect on initiation (31,33,40,47), whereas VP16 also strongly stimulates initiation. Tat is a unique activator because it is recruited to the transcription complex by binding to nascent RNA rather than to promoter DNA.…”

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Three Functional Classes of Transcriptional Activation Domains

Blau

Xiao

McCracken

et al. 1996

Molecular and Cellular Biology

258

263

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We have studied the abilities of different transactivation domains to stimulate the initiation and elongation (postinitiation) steps of RNA polymerase II transcription in vivo. Nuclear run-on and RNase protection analyses revealed three classes of activation domains: Sp1 and CTF stimulated initiation (type I); human immunodeficiency virus type 1 Tat fused to a DNA binding domain stimulated predominantly elongation (type IIA); and VP16, p53, and E2F1 stimulated both initiation and elongation (type IIB). A quadruple point mutation of VP16 converted it from a type IIB to a type I activator. Type I and type IIA activators synergized with one another but not with type IIB activators. This observation implies that synergy can result from the concerted action of factors stimulating two different steps in transcription: initiation and elongation. The functional differences between activators may be explained by the different contacts they make with general transcription factors. In support of this idea, we found a correlation between the abilities of activators, including Tat, to stimulate elongation and their abilities to bind TFIIH.Stimulation of eukaryotic gene expression requires sequence-specific factors with DNA binding domains and activation domains that interact with the general transcription factors (GTFs) and recruit RNA polymerase II (pol II) to the promoter (3, 65). In vivo the transcriptionally active form of pol II is probably a holoenzyme complex which contains a number of the GTFs as well as other polypeptides (36). Different activation domains interact with different GTFs, including TFIIB (44), TFIID (17,19,63), and TFIIF (76). These interactions are thought to recruit, stabilize, and/or modify the activity of the pol II holoenzyme.We recently demonstrated that the activation domains of p53, VP16, and E2F1 bind directly to TFIIH (50a, 72). TFIIH is a multisubunit factor, different forms of which are required for both transcription and nucleotide excision repair of DNA (9). TFIIH is the only GTF which has enzymatic activities: it has two helicase subunits and a cyclin-dependent protein kinase subunit which phosphorylates the pol II large-subunit C-terminal domain (CTD) (12,52,58,60). Both of these enzymatic activities are implicated in steps in transcription which occur shortly after initiation of the RNA chain. First, a helicase is required for efficient formation of open complexes and for promoter clearance on linear templates in vitro (20). Second, when paused polymerases resume elongation on several Drosophila genes in vivo, the CTD becomes phosphorylated, suggesting a possible role for TFIIH kinase in regulating elongation (50, 70). Furthermore, inhibitors of the TFIIH kinase inhibit elongation under activated (74), but not basal (57), transcription conditions.In vivo, rate-limiting steps after initiation have been well documented for a number of genes. For example, polymerases stall 20 to 40 bases downstream of the start sites in the Drosophila hsp70 and human c-myc genes (38, 51, 64) and terminate...

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HIV-1 Tat Overcomes Inefficient Transcriptional Elongation in Vitro

Cited by 60 publications

References 0 publications

The Tat Protein of Human Immunodeficiency Virus Type 1 Is a Substrate and Inhibitor of the Interferon-induced, Virally Activated Protein Kinase, PKR

The Tat Protein of Human Immunodeficiency Virus Type 1 Is a Substrate and Inhibitor of the Interferon-induced, Virally Activated Protein Kinase, PKR

RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo

Three Functional Classes of Transcriptional Activation Domains

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