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Query: UNIPROT:P23193 (transcription elongation factor)
 739 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Important progress in the understanding of elongation control by RNA polymerase II (RNAPII) has come from the recent identification of the positive transcription elongation factor b (P-TEFb) and the demonstration that this factor is a protein kinase that phosphorylates the carboxyl-terminal domain (CTD) of the RNAPII largest subunit. The P-TEFb complex isolated from mammalian cells contains a catalytic subunit (CDK9), a cyclin subunit (cyclin T1 or cyclin T2), and additional, yet unidentified, polypeptides of unknown function. To identify additional factors involved in P-TEFb function we performed a yeast two-hybrid screen using CDK9 as bait and found that cyclin K interacts with CDK9 in vivo. Biochemical analyses indicate that cyclin K functions as a regulatory subunit of CDK9. The CDK9-cyclin K complex phosphorylated the CTD of RNAPII and functionally substituted for P-TEFb comprised of CDK9 and cyclin T in in vitro transcription reactions.
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PMID:Cyclin K functions as a CDK9 regulatory subunit and participates in RNA polymerase II transcription. 1057 12

The human immunodeficiency virus 1 (HIV-1) Tat protein activates transcriptional elongation by recruiting the positive transcription elongation factor (pTEFb) complex to the TAR RNA element, which is located at the 5' extremity of all viral transcripts [1-3]. Tat also associates in vitro and in vivo with the transcriptional coactivator p300/CBP [4-6]. This association has been proposed to recruit the histone acetyltransferase (HAT) activity of p300 to the integrated HIV-1 promoter. We have observed that the purified p300 HAT domain acetylates recombinant Tat proteins in vitro and that Tat is acetylated in vivo. The major targets of acetylation by p300 are lysine residues (Lys50 and Lys51) in the arginine-rich motif (ARM) used by Tat to bind RNA and for nuclear import. Mutation of these residues in full-length recombinant Tat blocked its acetylation in vitro. Furthermore, mutation of these lysine residues to arginine markedly decreased the synergistic activation of he HIV promoter by Tat and p300 or by Tat and cyclin T1. These results demonstrate that acetylation of Tat by p300/CBP is important for its transcriptional activation of the HIV promoter.
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PMID:Acetylation of the HIV-1 Tat protein by p300 is important for its transcriptional activity. 1060 94

Tat activation of HIV-1 transcription is mediated by human transcription elongation factor P-TEFb, which interacts with Tat and phosphorylates the C-terminal domain of RNA polymerase II. The catalytic subunit of the P-TEFb complex, Cdk9, has been shown to interact with cyclin T and several other proteins of unknown identity. Consequently, the exact subunit composition of active P-TEFb has not been determined. Here we report the affinity purification and identification of the Cdk9-associated proteins. In addition to forming a heterodimer with cyclin T1, Cdk9 interacted with the molecular chaperone Hsp70 or a kinase-specific chaperone complex, Hsp90/Cdc37, to form two separate chaperone-Cdk9 complexes. Although the Cdk9/cyclin T1 dimer was exceptionally stable and produced slowly in the cell, free and unprotected Cdk9 appeared to be degraded rapidly. Several lines of evidence indicate the heterodimer of Cdk9/cyclin T1 to be the mature, active form of P-TEFb responsible for phosphorylation of the C-terminal domain of RNA polymerase II interaction with the Tat activation domain, and mediation of Tat activation of HIV-1 transcription. Pharmacological inactivation of Hsp90/Cdc37 function by geldanamycin revealed an essential role for the chaperone-Cdk9 complexes in generation of Cdk9/cyclin T1. Our data suggest a previously unrecognized chaperone-dependent pathway involving the sequential actions of Hsp70 and Hsp90/Cdc37 in the stabilization/folding of Cdk9 as well as the assembly of an active Cdk9/cyclin T1 complex responsible for P-TEFb-mediated Tat transactivation.
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PMID:Requirement for a kinase-specific chaperone pathway in the production of a Cdk9/cyclin T1 heterodimer responsible for P-TEFb-mediated tat stimulation of HIV-1 transcription. 1061 16

AIDS and the bare lymphocyte syndrome (BLS) are severe combined immunodeficiencies. BLS results from mutations in genes that regulate the expression of class II major histocompatibility (MHC II) determinants. One of these is the class II transactivator (CIITA). HIV and its transcriptional transactivator (Tat) also block the expression of MHC II genes. By binding to the same surface in the cyclin T1, which together with CDK9 forms the positive transcription elongation factor b (P-TEFb) complex, Tat inhibits CIITA. CIITA can also activate transcription when tethered artificially to RNA. Moreover, a dominant-negative CDK9 protein inhibits the activity of MHC II promoters. Thus, CIITA is a novel cellular coactivator that binds to P-TEFb for the expression of its target genes.
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PMID:Tat competes with CIITA for the binding to P-TEFb and blocks the expression of MHC class II genes in HIV infection. 1066 6

Transcriptional transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter element by the essential viral Tat protein requires recruitment of positive transcription elongation factor b (P-TEFb) to the viral TAR RNA target. The recruitment of P-TEFb, which has been proposed to be necessary and sufficient for activation of viral gene expression, is mediated by the highly cooperative interaction of Tat and cyclin T1, an essential component of P-TEFb, with the HIV-1 TAR element. Species, such as rodents, that encode cyclin T1 variants that are unable to support TAR binding by the Tat-cyclin T1 heterodimer are also unable to support HIV-1 Tat function. In contrast, we here demonstrate that the bovine immunodeficiency virus (BIV) Tat protein is fully able to bind to BIV TAR both in vivo and in vitro in the absence of any cellular cofactor. Nevertheless, BIV Tat can specifically recruit cyclin T1 to the BIV TAR element, and this recruitment is as essential for BIV Tat function as it is for HIV-1 Tat activity. However, because the cyclin T1 protein does not contribute to TAR binding, BIV Tat is able to function effectively in cells from several species that do not support HIV-1 Tat function. Thus, BIV Tat, while apparently dependent on the same cellular cofactor as the Tat proteins encoded by other lentiviruses, is nevertheless unique in terms of the mechanism used to recruit the BIV Tat-cyclin T1 complex to the viral LTR promoter.
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PMID:Functional differences between human and bovine immunodeficiency virus Tat transcription factors. 1077 3

Transcriptional transactivators (Tat) from many lentiviruses interact with their cognate transactivation response RNA structures (TAR) to increase rates of elongation rather than initiation of transcription. For several of them, the complex of Tat and a species-specific cyclin T1 must be formed before the binding to TAR can occur with high affinity and specificity. In sharp contrast, Tat from the bovine immunodeficiency virus (BIV) binds to its TAR without the help of the cyclin T1. This binding depends on the upper stem and 5' bulge, but not the central loop in TAR. Moreover, cyclins T1 from different species can mediate effects of this Tat in cells. Unlike the situation with other lentiviruses, Tat transactivation can be rescued simply by linking a heterologous promoter to TAR in permissive cells. Thus, lentiviruses have evolved different strategies to recruit Tat and the positive transcription elongation factor b to their promoters, and interactions between Tat and TAR are independent from those between Tat and the cyclin T1 in BIV.
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PMID:Binding of Tat to TAR and recruitment of positive transcription elongation factor b occur independently in bovine immunodeficiency virus. 1084 86

Tat stimulation of human immunodeficiency virus type 1 (HIV-1) transcription requires Tat-dependent recruitment of human positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter and the formation on the trans-acting response element (TAR) RNA of a P-TEFb-Tat-TAR ternary complex. We show here that the P-TEFb heterodimer of Cdk9-cyclin T1 is intrinsically incapable of forming a stable complex with Tat and TAR due to two built-in autoinhibitory mechanisms in P-TEFb. Both mechanisms exert little effect on the P-TEFb-Tat interaction but prevent the P-TEFb-Tat complex from binding to TAR RNA. The first autoinhibition arises from the unphosphorylated state of Cdk9, which establishes a P-TEFb conformation unfavorable for TAR recognition. Autophosphorylation of Cdk9 overcomes this inhibition by inducing conformational changes in P-TEFb, thereby exposing a region in cyclin T1 for possible TAR binding. An intramolecular interaction between the N- and C-terminal regions of cyclin T1 sterically blocks the P-TEFb-TAR interaction and constitutes the second autoinhibitory mechanism. This inhibition is relieved by the binding of the C-terminal region of cyclin T1 to the transcription elongation factor Tat-SF1 and perhaps other cellular factors. Upon release from the intramolecular interaction, the C-terminal region also interacts with RNA polymerase II and is required for HIV-1 transcription, suggesting its role in bridging the P-TEFb-Tat-TAR complex and the basal elongation apparatus. These data reveal novel control mechanisms for the assembly of a multicomponent transcription elongation complex at the HIV-1 promoter.
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PMID:Relief of two built-In autoinhibitory mechanisms in P-TEFb is required for assembly of a multicomponent transcription elongation complex at the human immunodeficiency virus type 1 promoter. 1091 73

Human immunodeficiency virus, type 1 (HIV-1), Tat activates elongation of RNA polymerase II transcription at the HIV-1 promoter through interaction with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex, P-TEFb. Binding of Tat to CycT1 induces cooperative binding of the P-TEFb complex onto nascent HIV-1 TAR RNA. Here the specific interaction between Tat protein, human cyclin T1, and HIV-1 TAR RNA was analyzed by fluorescence resonance energy transfer, using fluorescein-labeled TAR RNA and a rhodamine-labeled Tat protein synthesized through solid-phase chemistry. We find that CycT1 remodels the structure of Tat to enhance its affinity for TAR RNA and that TAR RNA further enhances the interaction between Tat and CycT1. We conclude that TAR RNA nucleates the formation of the Tat.P-TEFb complex through an induced fit mechanism.
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PMID:HIV-1 TAR RNA enhances the interaction between Tat and cyclin T1. 1094 37

Human immunodeficiency virus type 1 (HIV-1) Tat interacts with cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote transcription elongation. We show here that Tat also stimulates phosphorylation of affinity-purified core RNA polymerase II and glutathione S-transferase-C-terminal-domain substrates by CycT1-CDK9, but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant Tat-P-TEFb complexes with ATP enhanced binding to TAR RNA dramatically, and the C-terminal half of CycT1 masked binding of Tat to TAR RNA in the absence of ATP. ATP incubation lead to autophosphorylation of CDK9 at multiple C-terminal Ser and Thr residues, and full-length CycT1 (amino acids 728) [CycT1(1-728)], but not truncated CycT1(1-303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1-303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1-303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat-P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.
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PMID:CDK9 autophosphorylation regulates high-affinity binding of the human immunodeficiency virus type 1 tat-P-TEFb complex to TAR RNA. 1095 91

Equine infectious anemia virus (EIAV) activates transcription via a Tat protein, a TAR element, and the equine elongation factor positive transcription elongation factor b (P-TEFb). In human cells, EIAV Tat (eTat) can inhibit the ability of human immunodeficiency virus type 1 (HIV-1) Tat (hTat) to activate transcription from the HIV-1 long terminal repeat, demonstrating that EIAV Tat can interact nonproductively with human P-TEFb. To study the mechanism of EIAV Tat and HIV-1 Tat activation, we developed an in vitro elongation assay that recapitulates EIAV Tat-mediated inhibition of HIV-1 Tat trans-activation. We found that eTat specifically inhibits activation of elongation by HIV-1 Tat while having no effect on basal transcription elongation. The competitive inhibition of hTat activation was reversed by an activity present in HeLa cell nuclear extracts, most likely a form of P-TEFb. Recombinant P-TEFb (cyclin T1 and CDK9) overcame the inhibition of transcription by eTat but in a nonspecific manner. EIAV Tat affinity chromatography was used to purify the activity present in nuclear extract that was capable of reversing eTat inhibition. We characterized the protein components of this activity, which include cyclin T1, CDK9, Tat-SF1, and at least three unidentified proteins. These data suggest that additional factors are involved in the mechanism of Tat activation.
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PMID:An in vitro transcription system that recapitulates equine infectious anemia virus tat-mediated inhibition of human immunodeficiency virus type 1 Tat activity demonstrates a role for positive transcription elongation factor b and associated proteins in the mechanism of Tat activation. 1096 78


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