Tat Transactivation: A Model for the Regulation of Eukaryotic Transcriptional Elongation

Taube, Ran; Fujinaga, Koh; Wimmer, J; Barborič, Matjaž; Bm, Peterlin

doi:10.1006/viro.1999.9944

Cited by 117 publications

(111 citation statements)

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“…The second plasmid target, pG6SpCAT, contains six UASs and three Sp1 binding sites placed in front of the E1b TATA promoter and the CAT gene (Fig. 1A, pG6SpCAT) (45). The third plasmid target, pG6TARCAT, contains six UASs positioned upstream of the B sites in the human immunodeficiency virus type 1 long terminal repeat, which also transcribes the transactivation response (TAR) RNA stem loop and the CAT gene (45).…”

Section: Resultsmentioning

confidence: 99%

“…The third plasmid target, pG6TARCAT, contains six UASs positioned upstream of the B sites in the human immunodeficiency virus type 1 long terminal repeat, which also transcribes the transactivation response (TAR) RNA stem loop and the CAT gene (45). Three Sp1 sites load and position RNAPII on these two promoters, but they require an enhancer to recruit P-TEFb for the transition to the elongation phase of transcription and the expression of the CAT gene (45). We used these plasmid targets to determine whether AIRE stimulates the initiation and/or elongation of transcription.…”

Section: Resultsmentioning

confidence: 99%

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AIRE Recruits P-TEFb for Transcriptional Elongation of Target Genes in Medullary Thymic Epithelial Cells

Oven

Brdičková

Kohoutek

et al. 2007

Molecular and Cellular Biology

141

131

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AIRE is a transcriptional activator that directs the ectopic expression of many tissue-specific genes in medullary thymic epithelial cells, which plays an important role in the negative selection of autoreactive T cells. However, its mechanism of action remains poorly understood. In this study, we found that AIRE regulates the step of elongation rather than initiation of RNA polymerase II. For these effects, AIRE bound and recruited P-TEFb to target promoters in medullary thymic epithelial cells. In these cells, AIRE activated the ectopic transcription of insulin and salivary protein 1 genes. Indeed, by chromatin immunoprecipitation, we found that RNA polymerase II was already engaged on these promoters but was unable to elongate in the absence of AIRE. Moreover, the genetic inactivation of cyclin T1 from P-TEFb abolished the transcription of AIRE-responsive genes and led to lymphocytic infiltration of lacrimal and salivary glands in the CycT1 ؊/؊ mouse. Our findings reveal critical steps by which AIRE regulates the transcription of genes that control central tolerance in the thymus.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

AIRE Recruits P-TEFb for Transcriptional Elongation of Target Genes in Medullary Thymic Epithelial Cells

Oven

Brdičková

Kohoutek

et al. 2007

Molecular and Cellular Biology

141

131

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“…H IV-1 encodes a transcriptional activator protein, Tat, which is expressed early in the viral life cycle and is essential for viral gene expression, replication, and pathogenesis (1)(2)(3). Tat enhances processivity of RNA polymerase II (pol II) elongation complexes that initiate in the HIV long terminal repeat region.…”

mentioning

confidence: 99%

TAR RNA loop: A scaffold for the assembly of a regulatory switch in HIV replication

Richter

Ping²,

Rana

2002

Proc. Natl. Acad. Sci. U.S.A.

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Replication of HIV requires the Tat protein, which activates elongation of RNA polymerase II transcription at the HIV-1 promoter by interacting with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex b (P-TEFb). The transactivation domain of Tat binds directly to the CycT1 subunit of P-TEFb and induces loop sequence-specific binding of P-TEFb onto nascent HIV-1 trans-activation responsive region (TAR) RNA. We used systematic RNA-protein photocross-linking, Western blot analysis, and protein footprinting to show that residues 252-260 of CycT1 interact with one side of the TAR RNA loop and enhance interaction of Tat residue K50 to the other side of the loop. Our results show that TAR RNA provides a scaffold for two protein partners to bind and assemble a regulatory switch in HIV replication. RNA-mediated assembly of RNA-protein complexes could be a general mechanism for stable ribonucleoprotein complex formation and a key step in regulating other cellular processes and viral replication.H IV-1 encodes a transcriptional activator protein, Tat, which is expressed early in the viral life cycle and is essential for viral gene expression, replication, and pathogenesis (1-3). Tat enhances processivity of RNA polymerase II (pol II) elongation complexes that initiate in the HIV long terminal repeat region. In nuclear extracts, HIV-1 Tat associates tightly with the CDK9-containing positive transcription elongation factor complex b, P-TEFb (4-6). Recent studies indicate that Tat binds directly through its transactivation domain to the cyclin T1 (CycT1) subunit of the P-TEFb complex and induces loop sequence-specific binding of the P-TEFb complex to trans-activation responsive region (TAR) RNA (7-9).Recruitment of P-TEFb to TAR has been proposed to be both necessary and sufficient for activating transcription elongation from the HIV-1 long terminal repeat promoter (10). Neither CycT1 nor the P-TEFb complex bind TAR RNA in the absence of Tat; thus TAR binding is highly cooperative for both 9). In the C-terminal boundary of the CycT1 cyclin domain, Tat appears to contact residues that are not critical for CycT1 binding to CDK9 (8,(11)(12)(13)(14)(15). Mutagenesis studies showed that the CycT1 sequence containing amino acids 1-303 was sufficient to form complexes with Tat-TAR and CDK9 (8,(11)(12)(13)(14)(15). Recent fluorescence resonance energy-transfer studies using fluorescein-labeled TAR RNA and a rhodamine-labeled Tat protein showed that CycT1 remodels the structure of Tat to enhance its affinity for TAR RNA, and that TAR RNA further enhances interaction between Tat and CycT1 (16).The mechanism by which CycT1 induces loop sequence-specific binding of the P-TEFb complex onto nascent HIV-1 TAR RNA is not understood presently. Does CycT1 interact directly with the TAR loop or merely reorganize Tat structure to bind the loop residues? Does Tat bind TAR loop in the presence of CycT1? What regions of CycT1 and Tat interact directly with the TAR loop sequence? Does phosphorylation of P-TEFb chan...

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“…Tat activates transcription of viral genes through direct binding to CycT1 and promotes cooperative binding to TAR, a stem-loop RNA structure formed at the 5′ end of the nascent viral transcript. 23,24 This results in activation of viral gene expression at the level of transcript elongation. A critical cysteine (Cys261) of human CycT1 is thought to form an intermolecular zinc-finger with cysteines from Tat.…”

Section: Introductionmentioning

confidence: 99%

Cyclin Box Structure of the P-TEFb Subunit Cyclin T1 Derived from a Fusion Complex with EIAV Tat

Anand¹,

Schulte²,

Fujinaga³

et al. 2007

Journal of Molecular Biology

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SummaryThe positive transcription elongation factor b (P-TEFb) is an essential regulator of viral gene expression during the life cycle of HIV-1. Its Cyclin T1 subunit forms a ternary complex with the viral Tat protein and the TAR RNA element thereby activating cyclin dependent kinase 9 (Cdk9), which stimulates transcription at the level of chain elongation. We report the structure of the cyclin box domain of human Cyclin T1 at a resolution of 2.67 Å. The structure was obtained by crystallographic analysis of a fusion protein composed of Cyclin T1 linked to the transactivator protein Tat from equine infectious anemia virus (EIAV), which is functionally and structurally related to HIV-1 Tat. The conserved cyclin box domain of Cyclin T1 exhibits structural features for interaction with physiological binding partners such as Cdk9. A recognition site for Cdk/Cyclin substrates is partly covered by a Cyclin T-specific insert, suggesting specific interactions with regulatory factors. The previously identified Tat/TAR recognition motif (TRM) forms a C-terminal helix that is partly occluded in the cyclin box repeat interface, while cysteine 261 is accessible to form an intermolecular zinc finger with Tat. Residues of the TRM contribute to a positively charged groove that may directly attract RNA molecules. The EIAV Tat protein instead appeared undefined from the electron density map suggesting that it is highly disordered. Functional experiments confirmed the TAR binding properties of the fusion protein and suggested residues on the second cyclin box repeat to contribute to Tat stimulated transcription.

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Tat Transactivation: A Model for the Regulation of Eukaryotic Transcriptional Elongation

Cited by 117 publications

References 108 publications

AIRE Recruits P-TEFb for Transcriptional Elongation of Target Genes in Medullary Thymic Epithelial Cells

AIRE Recruits P-TEFb for Transcriptional Elongation of Target Genes in Medullary Thymic Epithelial Cells

TAR RNA loop: A scaffold for the assembly of a regulatory switch in HIV replication

Cyclin Box Structure of the P-TEFb Subunit Cyclin T1 Derived from a Fusion Complex with EIAV Tat

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