RESUMO
Elegant studies by Hasler et al. (2020) and Wang et al. (2020) uncover a novel role of LARP7 in facilitating the 2'-O-methylation of the spliceosomal U6 snRNA, which is functionally required for fidelity of pre-mRNA splicing and development of male germ cells.
Assuntos
Splicing de RNA , RNA Nuclear Pequeno , Animais , Sequência de Bases , Masculino , Camundongos , Conformação de Ácido Nucleico , EspermatogêneseRESUMO
DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill defined. Here, we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates RNA polymerase II (Pol II) transcription and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb) via its release from the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP). This is mediated by activation of p38MAPK, which triggers enhanced binding of RBM7 with core subunits of 7SK snRNP. In turn, P-TEFb relocates to chromatin to induce transcription of short units, including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with the axis of RBM7 and P-TEFb provokes cellular hypersensitivity to DNA-damage-inducing agents due to activation of apoptosis. Our work uncovers the importance of stress-dependent stimulation of Pol II pause release, which enables a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult.
Assuntos
Fator B de Elongação Transcricional Positiva/genética , RNA Polimerase II/genética , Proteínas de Ligação a RNA/genética , Transcrição Gênica , Apoptose/genética , Sobrevivência Celular/genética , Dano ao DNA/genética , Células HEK293 , Humanos , RNA Longo não Codificante/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
P-TEFb and CDK12 facilitate transcriptional elongation by RNA polymerase II. Given the prominence of both kinases in cancer, gaining a better understanding of their interplay could inform the design of novel anti-cancer strategies. While down-regulation of DNA repair genes in CDK12-targeted cancer cells is being explored therapeutically, little is known about mechanisms and significance of transcriptional induction upon inhibition of CDK12. We show that selective targeting of CDK12 in colon cancer-derived cells activates P-TEFb via its release from the inhibitory 7SK snRNP. In turn, P-TEFb stimulates Pol II pause release at thousands of genes, most of which become newly dependent on P-TEFb. Amongst the induced genes are those stimulated by hallmark pathways in cancer, including p53 and NF-κB. Consequently, CDK12-inhibited cancer cells exhibit hypersensitivity to inhibitors of P-TEFb. While blocking P-TEFb triggers their apoptosis in a p53-dependent manner, it impedes cell proliferation irrespective of p53 by preventing induction of genes downstream of the DNA damage-induced NF-κB signaling. In summary, stimulation of Pol II pause release at the signal-responsive genes underlies the functional dependence of CDK12-inhibited cancer cells on P-TEFb. Our study establishes the mechanistic underpinning for combinatorial targeting of CDK12 with either P-TEFb or the induced oncogenic pathways in cancer.
Assuntos
Fator B de Elongação Transcricional Positiva , RNA Polimerase II , Neoplasias/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Supressora de Tumor p53/genética , Humanos , Linhagem Celular TumoralRESUMO
Positive transcription elongation factor b (P-TEFb) is the crucial player in RNA polymerase II (Pol II) pause release that has emerged as a promising target in cancer. Because single-agent therapy may fail to deliver durable clinical response, targeting of P-TEFb shall benefit when deployed as a combination therapy. We screened a comprehensive oncology library and identified clinically relevant antimetabolites and Mouse double minute 2 homolog (MDM2) inhibitors as top compounds eliciting p53-dependent death of colorectal cancer cells in synergy with selective inhibitors of P-TEFb. While the targeting of P-TEFb augments apoptosis by anti-metabolite 5-fluorouracil, it switches the fate of cancer cells by the non-genotoxic MDM2 inhibitor Nutlin-3a from cell-cycle arrest to apoptosis. Mechanistically, the fate switching is enabled by the induction of p53-dependent pro-apoptotic genes and repression of P-TEFb-dependent pro-survival genes of the PI3K-AKT signaling cascade, which stimulates caspase 9 and intrinsic apoptosis pathway in BAX/BAK-dependent manner. Finally, combination treatments trigger apoptosis of cancer cell spheroids. Together, co-targeting of P-TEFb and suppressors of intrinsic apoptosis could become a viable strategy to eliminate cancer cells.
Assuntos
Apoptose , Fator B de Elongação Transcricional Positiva , Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53 , Linhagem Celular Tumoral , Sobrevivência Celular , Fosfatidilinositol 3-Quinases/metabolismo , Fator B de Elongação Transcricional Positiva/antagonistas & inibidores , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , HumanosRESUMO
Release of RNA polymerase II (Pol II) from promoter-proximal pausing has emerged as a critical step regulating gene expression in multicellular organisms. The transition of Pol II into productive elongation requires the kinase activity of positive transcription elongation factor b (P-TEFb), which is itself under a stringent control by the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. Here, we provide an overview on stimulating Pol II pause release by P-TEFb and on sequestering P-TEFb into 7SK snRNP. Furthermore, we highlight mechanisms that govern anchoring of 7SK snRNP to chromatin as well as means that release P-TEFb from the inhibitory complex, and propose a unifying model of P-TEFb activation on chromatin. Collectively, these studies shine a spotlight on the central role of RNA binding proteins (RBPs) in directing the inhibition and activation of P-TEFb, providing a compelling paradigm for controlling Pol II transcription with a non-coding RNA.
Assuntos
Fator B de Elongação Transcricional Positiva/metabolismo , RNA Polimerase II/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Elongação da Transcrição Genética , Animais , Cromatina/metabolismo , Humanos , Modelos BiológicosRESUMO
Influenza NS1 protein is an important virulence factor that is capable of binding double-stranded (ds) RNA and inhibiting dsRNA-mediated host innate immune responses. Here we show that NS1 can also bind cellular dsDNA. This interaction prevents loading of transcriptional machinery to the DNA, thereby attenuating IAV-mediated expression of antiviral genes. Thus, we identified a previously undescribed strategy, by which RNA virus inhibits cellular transcription to escape antiviral response and secure its replication.
Assuntos
DNA/metabolismo , Transcrição Gênica/fisiologia , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Humanos , Vírus da Influenza A/fisiologia , Ligação Proteica , Proteínas não Estruturais Virais/fisiologia , Replicação ViralRESUMO
The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.
Assuntos
Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Reparo do DNA/genética , Mutação , Sequência de Aminoácidos , Western Blotting , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/química , Ciclinas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
The positive transcription elongation factor b (P-TEFb) regulates RNA polymerase II elongation. In cells, P-TEFb partitions between small active and larger inactive states. In the latter, HEXIM1 binds to 7SK snRNA and recruits as well as inactivates P-TEFb in the 7SK snRNP. Several stimuli can affect this P-TEFb equilibrium. In this study, we demonstrate that protein kinase C (PKC) phosphorylates the serine at position158 (S158) in HEXIM1. This phosphorylated HEXIM1 protein neither binds to 7SK snRNA nor inhibits P-TEFb. Phorbol esters or the engagement of the T cell antigen receptor, which activate PKC and the expression of the constitutively active (CA) PKCθ protein, which is found in T cells, inhibit the formation of the 7SK snRNP. All these stimuli increase P-TEFb-dependent transcription. In contrast, the kinase-negative PKCθ and the mutant HEXIM1 (S158A) proteins block effects of these PKC-activating stimuli. These results indicate that the phosphorylation of HEXIM1 by PKC represents a major regulatory step of P-TEFb activity in cells.
Assuntos
Fator B de Elongação Transcricional Positiva/metabolismo , Proteína Quinase C-delta/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Humanos , Células Jurkat , Dados de Sequência Molecular , NF-kappa B/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Receptores de Antígenos de Linfócitos T/imunologia , Serina/metabolismo , Fatores de TranscriçãoRESUMO
Promoter-proximal pausing of RNAPII coincides with the formation of the cap structure at the 5' end of pre-mRNA, which is bound by the cap-binding protein complex (CBC). Although the positive transcription elongation factor b (P-TEFb) stimulates the release of RNAPII from pausing and promotes transcription elongation and alternative splicing by phosphorylating the RNAPII C-terminal domain at Ser2 (S2-P RNAPII), it is unknown whether CBC facilitates these events. In this study, we report that CBC interacts with P-TEFb and transcriptionally engaged RNAPII and is globally required for optimal levels of S2-P RNAPII. Quantitative nascent RNA immunoprecipitation and ChIP experiments reveal that depletion of CBC attenuates HIV-1 Tat transactivation and impedes transcription elongation of investigated CBC-dependent endogenous genes by decreasing the levels of P-TEFb and S2-P RNAPII, leading to accumulation of RNAPII in the body of these genes. Finally, CBC is essential for the promotion of alternative splicing through facilitating P-TEFb, S2-P RNAPII, and splicing factor 2/alternative splicing factor occupancy at a splicing minigene. These findings disclose a vital role of CBC in connecting pre-mRNA capping to transcription elongation and alternative splicing via P-TEFb.
Assuntos
Processamento Alternativo/fisiologia , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas de Ligação ao Cap de RNA/metabolismo , Precursores de RNA/biossíntese , Transcrição Gênica/fisiologia , Células HeLa , Humanos , Fator B de Elongação Transcricional Positiva/genética , Proteínas de Ligação ao Cap de RNA/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genéticaRESUMO
Eukaryotic gene expression is commonly controlled at the level of RNA polymerase II (RNAPII) pausing subsequent to transcription initiation. Transcription elongation is stimulated by the positive transcription elongation factor b (P-TEFb) kinase, which is suppressed within the 7SK small nuclear ribonucleoprotein (7SK snRNP). However, the biogenesis and functional significance of 7SK snRNP remain poorly understood. Here, we report that LARP7, BCDIN3, and the noncoding 7SK small nuclear RNA (7SK) are vital for the formation and stability of a cell stress-resistant core 7SK snRNP. Our functional studies demonstrate that 7SK snRNP is not only critical for controlling transcription elongation, but also for regulating alternative splicing of pre-mRNAs. Using a transient expression splicing assay, we find that 7SK snRNP disintegration promotes inclusion of an alternative exon via the increased occupancy of P-TEFb, Ser2-phosphorylated (Ser2-P) RNAPII, and the splicing factor SF2/ASF at the minigene. Importantly, knockdown of larp7 or bcdin3 orthologues in zebrafish embryos destabilizes 7SK and causes severe developmental defects and aberrant splicing of analyzed transcripts. These findings reveal a key role for P-TEFb in coupling transcription elongation with alternative splicing, and suggest that maintaining core 7SK snRNP is essential for vertebrate development.
Assuntos
Processamento Alternativo , Fator B de Elongação Transcricional Positiva/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Motivos de Aminoácidos , Animais , Regulação da Expressão Gênica no Desenvolvimento , Variação Genética , Modelos Genéticos , Fosforilação , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Ribonuclease Pancreático/metabolismo , Transcrição Gênica , Vertebrados/fisiologia , Peixe-ZebraRESUMO
Promoter-proximal pausing of RNA polymerase II (RNAPII) across the genome has renewed our attention to the early transcriptional events that control the establishment of pausing and the release of RNAPII into a productive transcription elongation. Here, we review our current understanding of the transcriptional cycle by RNAPII with a particular emphasis on the mechanisms that stimulate transcription elongation and cotranscriptional pre-mRNA splicing through an essential transcriptional kinase, the positive transcription elongation factor b (P-TEFb). We illustrate that by targeting a limited set of transcription elongation factors and paused RNAPII molecule during a promoter-proximal phase of transcription, P-TEFb unleashes an extensive crosstalk between transcription apparatus, RNA processing factors and chromatin for optimal production of mRNA.
Assuntos
Fator B de Elongação Transcricional Positiva/metabolismo , Precursores de RNA/genética , Splicing de RNA/genética , Transcrição Gênica , Animais , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismoRESUMO
Hexamethylene bisacetamide (HMBA) is a potent inducer of cell differentiation and HIV production in chronically infected cells. However, its mechanism of action remains poorly defined. In this study, we demonstrate that HMBA activates transiently the PI3K/Akt pathway, which leads to the phosphorylation of HEXIM1 and the subsequent release of active positive transcription elongation factor b (P-TEFb) from its transcriptionally inactive complex with HEXIM1 and 7SK small nuclear RNA (snRNA). As a result, P-TEFb is recruited to the HIV promoter to stimulate transcription elongation and viral production. Despite the continuous presence of HMBA, the released P-TEFb reassembles rapidly with 7SK snRNA and HEXIM1. In contrast, a mutant HEXIM1 protein that cannot be phosphorylated and released from P-TEFb and 7SK snRNA via the PI3K/Akt pathway antagonizes this HMBA-mediated induction of viral production. Thus, our studies reveal how HIV transcription is induced by HMBA and suggest how modifications in the equilibrium between active and inactive P-TEFb could contribute to cell differentiation.
Assuntos
Acetamidas/farmacologia , Antineoplásicos/farmacologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , HIV-1/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Relação Dose-Resposta a Droga , HIV-1/metabolismo , Humanos , Células Jurkat , Fatores de TranscriçãoRESUMO
Human immunodeficiency virus type 1 (HIV-1) transcriptional transactivator (Tat) recruits the positive transcription elongation factor b (P-TEFb) to the viral promoter. Consisting of cyclin dependent kinase 9 (Cdk9) and cyclin T1, P-TEFb phosphorylates RNA polymerase II and the negative transcription elongation factor to stimulate the elongation of HIV-1 genes. A major fraction of nuclear P-TEFb is sequestered into a transcriptionally inactive 7SK small nuclear ribonucleoprotein (snRNP) by the coordinated actions of the 7SK small nuclear RNA (snRNA) and hexamethylene bisacetamide (HMBA) induced protein 1 (HEXIM1). In this study, we demonstrate that Tat prevents the formation of and also releases P-TEFb from the 7SK snRNP in vitro and in vivo. This ability of Tat depends on the integrity of its N-terminal activation domain and stems from the high affinity interaction between Tat and cyclin T1, which allows Tat to directly displace HEXIM1 from cyclin T1. Furthermore, we find that in contrast to the Tat-independent activation of the HIV-1 promoter, Tat-dependent HIV-1 transcription is largely insensitive to the inhibition by HEXIM1. Finally, primary blood lymphocytes display a reduced amount of the endogenous 7SK snRNP upon HIV-1 infection. All these data are consistent with the model that Tat not only recruits but also increases the active pool of P-TEFb for efficient HIV-1 transcription.
Assuntos
Regulação Viral da Expressão Gênica , Produtos do Gene tat/metabolismo , HIV-1/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ligação Competitiva , Ciclina T , Ciclinas , Células HeLa , Humanos , Linfócitos/metabolismo , Linfócitos/virologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Fatores de Transcrição , Transcrição Gênica , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Transcriptional elongation of most eukaryotic genes by RNA polymerase II requires the kinase activity of the positive transcription elongation factor b (P-TEFb). The catalytically active P-TEFb complex becomes inactive when sequestered into the large complex by the cooperative actions of 7SK snRNA and HEXIM1. In this study, we report that HEXIM1 forms oligomers in cells. This oligomerization is mediated by its predicted coiled-coil region in the C-terminal domain and 7SK snRNA that binds a basic region within the central part of HEXIM1. Alanine-mutagenesis of evolutionary conserved leucines in the coiled-coil region and the digestion of 7SK snRNA by RNase A treatment prevent this oligomerization. Importantly, mutations of the N-terminal part of the coiled-coil region abrogate the ability of HEXIM1 to bind and inhibit P-TEFb. Finally, the formation of HEXIM1 oligomers via the C-terminal part of the coiled-coil or basic regions is critical for the inhibition of transcription. Our results suggest that two independent regions in HEXIM1 form oligomers to incorporate P-TEFb into the large complex and determine the inhibition of transcriptional elongation.
Assuntos
Fator B de Elongação Transcricional Positiva/antagonistas & inibidores , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Evolução Molecular , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Fatores de Transcrição , Transcrição GênicaRESUMO
A virus protein called Tat plays a dual role in HIV infection by regulating the expression of genes belonging to the virus and genes belonging to the host cells.
Assuntos
Infecções por HIV/virologia , HIV/fisiologia , Interações Hospedeiro-Patógeno , Replicação Viral , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Transcrição GênicaAssuntos
Produtos do Gene tat/genética , HIV/genética , Transcrição Gênica , HIV/enzimologia , Modelos Biológicos , Biologia Molecular/tendências , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fatores de Elongação da Transcrição/genética , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
The generation of messenger RNA (mRNA) in eukaryotes is achieved by transcription from the DNA template and pre-mRNA processing reactions of capping, splicing, and polyadenylation. Although RNA polymerase II (RNAPII) catalyzes the synthesis of pre-mRNA, it also serves as a principal coordinator of the processing reactions in the course of transcription. In this review, we focus on the interplay between transcription and cotranscriptional pre-mRNA maturation events, mediated by the recruitment of RNA processing factors to differentially phosphorylated C-terminal domain of Rbp1, the largest subunit of RNAPII. Furthermore, we highlight the bidirectional nature of the interplay by discussing the impact of RNAPII kinetics on pre-mRNA processing as well as how the processing events reach back to different phases of gene transcription.
Assuntos
Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Transcrição Gênica , Animais , Humanos , Precursores de RNA/genética , RNA Mensageiro/genéticaRESUMO
The HIV-1 Tat protein promotes viral transcription elongation by recruiting P-TEFb to RNA element TAR on the viral mRNA. Recent work from D'Orso and Frankel uncovers unexpected aspects of this process.
Assuntos
Regulação Viral da Expressão Gênica , Repetição Terminal Longa de HIV/genética , HIV-1/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Transcrição Gênica , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Modelos Genéticos , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Polimerase II/metabolismo , Ativação Transcricional/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Hexim1 is a cellular protein that associates with the positive transcription elongation factor b (P-TEFb) to regulate RNA polymerase II elongation of nascent mRNA transcripts. It directly binds to Cyclin T1 of P-TEFb and inhibits the kinase activity of Cdk9, leading to an arrest of transcription elongation. Here, we report the solution structure of the Cyclin T binding domain (TBD) of Hexim1 that forms a parallel coiled-coil homodimer composed of two segments and a preceding alpha helix that folds back onto the first coiled-coil unit. NMR titration, fluorescence, and immunoprecipitation experiments revealed the binding interface to Cyclin T1, which covers a large surface on the first coiled-coil segment. Electrostatic interactions between an acidic patch on Hexim1 and positively charged residues of Cyclin T1 drive the complex formation that is confirmed by mutagenesis data on Hexim1 mediated transcription regulation in cells. Thus, our studies provide structural insights how Hexim1 recognizes the Cyclin T1 subunit of P-TEFb, which is a key step toward the regulation of transcription elongation.
Assuntos
Ciclinas/química , Ciclinas/metabolismo , Fator B de Elongação Transcricional Positiva/química , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação , Ciclina T , Ciclinas/genética , Dimerização , Células HeLa , Humanos , Modelos Moleculares , Mutação/genética , Ressonância Magnética Nuclear Biomolecular , Fator B de Elongação Transcricional Positiva/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Titulometria , Fatores de TranscriçãoRESUMO
The active form of the positive transcription elongation factor b (P-TEFb) consists of cyclin T and the kinase Cdk9. P-TEFb stimulates transcription by phosphorylating the C-terminal domain of RNA polymerase II. It becomes inactivated when associated in a tetrameric complex with the abundant 7SK small nuclear RNA and the recently identified protein Hexim1. In this study, we identified a stable and soluble C-terminal domain (residues 255-359) in Hexim1 of 12.5-kDa size that binds the cyclin boxes of Cyclin T1. Functional assays in HeLa cells showed that this cyclin T-binding domain (TBD) is required for the binding of Hexim1 to P-TEFb and inhibition of transcriptional activity in vivo. Analytical gel filtration and GST pull-down experiments revealed that both full-length Hexim1 and the TBD are homodimers. Isothermal titration calorimetry yielded a weak multimer for the TBD with a multimerization constant of 1.3 x 10(3) m. The binding affinity between the TBD and cyclin T1 was analyzed with fluorescence spectroscopy methods, using a dansyl-based fluorescence label at position G257C. Equilibrium fluorescence titration and stopped flow fast kinetics yield a dissociation constant of 1.2 mum. Finally, we tested the effect of the HIV-1 Tat protein on the cyclin T1-TBD complex formation. GST pull-down experiments and size exclusion chromatography exhibit a mutually exclusive binding of the two effectors to cyclin T1. Our data suggest a model where HIV-1 Tat competes with Hexim1 for cyclin T1 binding, thus releasing P-TEFb from the inactive complex to stimulate the transcription of HIV-1 gene expression.