HIV-1 TAR RNA Enhances the Interaction between Tat and Cyclin T1

Zhang, Jing; Tamilarasu, Natarajan; Hwang, Sangwon; Garber, Mitchell E.; Huq, Ikramul; Jones, Katherine A.; Rana, Tariq M.

doi:10.1074/jbc.m006804200

Cited by 71 publications

(60 citation statements)

References 35 publications

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“…In previous experiments, dissociation constants of 9.1 and 0.4 nM between the complex of Tat and CycT1-(1-303) in the absence and presence of TAR RNA were reported, respectively (30). Since these affinities to CycT1 are more than 100 times tighter compared with the dissociation constant between CycT1 and Hexim1-TBD identified here, it is surprising that Tat is not precipitated significantly better from solution than the TBD.…”

Section: Discussionsupporting

confidence: 55%

Identification of a Cyclin T-Binding Domain in Hexim1 and Biochemical Analysis of Its Binding Competition with HIV-1 Tat

Schulte

Czudnochowski

Barborič

et al. 2005

Journal of Biological Chemistry

101

120

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Section: Discussionsupporting

confidence: 55%

Identification of a Cyclin T-Binding Domain in Hexim1 and Biochemical Analysis of Its Binding Competition with HIV-1 Tat

Schulte

Czudnochowski

Barborič

et al. 2005

Journal of Biological Chemistry

101

120

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“…TAR RNA has been shown recently to strongly enhance the interaction between Tat and CycT1 (16). RNA-induced protein-protein interactions have been documented most clearly with the N protein, which binds to a site in the boxB RNA to mediate transcriptional antitermination (28)(29)(30)(31).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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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“…23). Binding of Tat to cyclin T1 induces co-operative binding of P-TEFb onto nascent TAR RNA (24), which in turn increases Tat affinity for TAR (25). Biochemically, Tat appears to contact residues in the amino terminus of cyclin T1, which are not essential for binding of cyclin T1 to CDK9, through its trans-activation domain (24, 26 -30).…”

mentioning

confidence: 99%

Visualization of in Vivo Direct Interaction between HIV-1 TAT and Human Cyclin T1 in Specific Subcellular Compartments by Fluorescence Resonance Energy Transfer

Marcello¹,

Cinelli²,

Ferrari³

et al. 2001

Journal of Biological Chemistry

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Human cyclin T1, a component of the P-TEFb kinase complex, was originally identified through its biochemical interaction with the Tat transactivator protein of human immunodeficiency virus type 1 (HIV-1). Current understanding suggests that binding of Tat to P-TEFb is required to promote efficient transcriptional elongation of viral RNAs. However, the dynamics and the subnuclear localization of this process are still largely unexplored in vivo. Here we exploit high resolution fluorescence resonance energy transfer (FRET) to visualize and quantitatively analyze the direct interaction between Tat and cyclin T1 inside the cells. We observed that cyclin T1 resides in specific subnuclear foci which are in close contact with nuclear speckles and that Tat determines its redistribution outside of these compartments. Consistent with this observation, strong FRET was observed between the two proteins both in the cytoplasm and in regions of the nucleus outside of cyclin T1 foci and overlapping with Tat localization. These results are consistent with a model by which Tat recruits cyclin T1 outside of the nuclear compartments where the protein resides to promote transcriptional activation.The human immunodeficiency virus type 1 (HIV-1) 1 transactivator protein Tat is a small polypeptide (86 -101 amino acids, according to the viral strains) essential for efficient transcription of viral genes. The protein is a highly unusual transcription factor since, at the HIV LTR promoter, it interacts with a cis-acting RNA element (trans-activation-responsive region, TAR) present at the 5Ј-end of each viral transcript (1).Through this interaction, Tat activates HIV-1 transcription by promoting the assembly of transcriptionally active complexes at the LTR by multiple protein-protein interactions (for a recent review, see Ref. 2). Over the last few years a number of cellular proteins have been reported to interact with Tat and to mediate or modulate its activity. These include general transcription factors, among which TBP, TAFII250, TFIIB, TFIIH (3-7); RNA polymerase II (8); transcription factor Sp1 (9); the transcriptional co-activators and histone acetyltransferases p300/CBP and P/CAF (10 -12); and the cyclin subunit of the positive transcription elongation factor complex (P-TEFb), cyclin T1 (13-16).The finding that Tat biochemically and functionally interacts with several cellular proteins raises some fundamental questions. Does Tat directly interact with its partners inside live cells? Which is the subcellular compartment of these interactions? Are they occurring simultaneously or consecutively? Some of these questions can be successfully addressed by taking advantage of fluorescence resonance energy transfer (FRET) measurements (17), allowing investigation of direct interaction of proteins labeled with optically matched fluorophores. FRET exploits radiationless energy transfer driven by dipole-dipole interaction occurring from a fluorophore (the donor) in the excited state to another fluorophore (the acceptor) when in close proximity...

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HIV-1 TAR RNA Enhances the Interaction between Tat and Cyclin T1

Cited by 71 publications

References 35 publications

Identification of a Cyclin T-Binding Domain in Hexim1 and Biochemical Analysis of Its Binding Competition with HIV-1 Tat

Identification of a Cyclin T-Binding Domain in Hexim1 and Biochemical Analysis of Its Binding Competition with HIV-1 Tat

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

Visualization of in Vivo Direct Interaction between HIV-1 TAT and Human Cyclin T1 in Specific Subcellular Compartments by Fluorescence Resonance Energy Transfer

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