Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The transition from abortive into productive elongation is proposed to be controlled by a positive transcription elongation factor b (P-TEFb) through phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Drosophila P-TEFb was identified recently as a cyclin-dependent kinase (CDK9) paired with a cyclin subunit (cyclin T). We demonstrate here the cloning of multiple cyclin subunits of human P-TEFb (T1 and T2). Cyclin T2 has two forms (T2a and T2b) because of alternative splicing. Both cyclin T1 and T2 are ubiquitously expressed. Immunoprecipitation and immunodepletion experiments carried out on HeLa nuclear extract (HNE) indicated that cyclin T1 and T2 were associated with CDK9 in a mutually exclusive manner and that almost all CDK9 was associated with either cyclin T1 or T2. Recombinant CDK9/cyclin T1, CDK9/cyclin T2a, and CDK9/cyclin T2b produced in Sf9 cells possessed DRB-sensitive kinase activity and functioned in transcription elongation in vitro. Either cyclin T1 or T2 was required to activate CDK9, and the truncation of the carboxyl terminus of the cyclin reduced, but did not eliminate, P-TEFb activity. Cotransfection experiments indicated that all three CDK9/cyclin combinations dramatically activated the CMV promoter.
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PMID:Identification of multiple cyclin subunits of human P-TEFb. 949 9

Positive transcription elongation factor b (P-TEFb) stimulates the transition from transcription initiation to productive elongation by phosphorylation of the C-terminal domain of RNA polymerase II. P-TEFb consists of the cyclin-dependent kinase Cdk9 and a T-type cyclin and is regulated by the small nuclear RNA 7SK and the coupling protein Hexim1 or Hexim2. In this study, we analyzed the tripartite protein-RNA complex formation between Hexim, Cyclin T and 7SK snRNA. Using isothermal titration calorimetry, we observed higher affinities for Cyclin T1-Hexim1 and Cyclin T2-Hexim2 complex formations compared with the interactions in reverse. Importin alpha, which is part of the Ran-mediated nuclear import pathway, bound Hexim1 and Hexim2 with dissociation constants of 2.0 and 0.5 muM, respectively. Furthermore, tripartite complex formations between Cyclin T, Hexim and Importin alpha showed the suitability of a collaborative nuclear import pathway for Cyclin T. Electrophoretic mobility shift assays using radioactively labelled full-length 7SK snRNA revealed a tight association of the RNA to Cyclin T1-Hexim1 with dissociation constants lower than 0.3 muM. Similar binding affinities were recorded for both Hexim orthologues to a 66-mer double-stranded 5' hairpin loop encompassing nucleotides 23-88 of 7SK, while a 39-mer fragment, resulting from different RNA folding predictions, did not bind as tightly. These results provide the molecular basis for the generation of a core complex for the inhibition of P-TEFb.
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PMID:Specificity of Hexim1 and Hexim2 complex formation with cyclin T1/T2, importin alpha and 7SK snRNA. 1988 59

Cyclin-dependent kinase 9 (Cdk9) is a serine-threonine kinase, involved in many cellular processes. The regulatory units of Cdk9 are the T family Cyclins (T1, T2) and Cyclin K. Cyclin T2 has two forms termed Cyclin T2a and Cyclin T2b that arise by an alternative splicing of the primary transcript. Upon induction of muscle differentiation, MyoD recruits Cdk9/Cyclin T2 on muscle-specific gene promoter sequences. This complex is able to phosphorylate the C-terminal domain of RNA polymerase II, enhancing MyoD function and promoting myogenic differentiation. This work focuses on the characterization of two murine Cyclin T2 isoforms and the evaluation of the role of Cdk9/Cyclin T2 complexes during the skeletal muscle differentiation. This study demonstrated a predominant expression of isoform b in all stages of differentiation. Moreover, both isoforms of Cyclin T2 are able to activate the myogenic program but Cyclin T2b has a predominant role, in particular during the latest stages.
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PMID:Activation and function of murine Cyclin T2A and Cyclin T2B during skeletal muscle differentiation. 2306 74

Productive transcription of the integrated HIV-1 provirus is restricted by cellular factors that inhibit RNA polymerase II elongation. The viral Tat protein overcomes this by recruiting a general elongation factor, P-TEFb, to the TAR RNA element that forms at the 5' end of nascent viral transcripts. P-TEFb exists in multiple complexes in cells, and its core consists of a kinase, Cdk9, and a regulatory subunit, either Cyclin T1 or Cyclin T2. Tat binds directly to Cyclin T1 and thereby targets the Cyclin T1/P-TEFb complex that phosphorylates the CTD of RNA polymerase II and the negative factors that inhibit elongation, resulting in efficient transcriptional elongation. P-TEFb is tightly regulated in cells infected by HIV-1-CD4+ T lymphocytes and monocytes/macrophages. A number of mechanisms have been identified that inhibit P-TEFb in resting CD4+ T lymphocytes and monocytes, including miRNAs that repress Cyclin T1 protein expression and dephosphorylation of residue Thr186 in the Cdk9 T-loop. These repressive mechanisms are overcome upon T cell activation and macrophage differentiation when the permissivity for HIV-1 replication is greatly increased. This review will summarize what is currently known about mechanisms that regulate P-TEFb and how this regulation impacts HIV-1 replication and latency.
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PMID:Making a Short Story Long: Regulation of P-TEFb and HIV-1 Transcriptional Elongation in CD4+ T Lymphocytes and Macrophages. 2483 49

Positive transcription elongation factor b (P-TEFb) is a RNA polymerase II carboxyl-terminal domain (Pol II CTD) kinase that phosphorylates Ser2 of the CTD and promotes the elongation phase of transcription. Despite the fact that P-TEFb has role in many cellular processes, the role of this kinase complex remains to be understood in mammalian early developmental events. In this study, using immunocytochemical analyses, we found that the P-TEFb components, CDK9, Cyclin T1 and Cyclin T2 were localized to nuclear speckles, as well as in nucleolar-like bodies in pig germinal vesicle oocytes. Using nascent RNA labeling and small molecule inhibitors, we showed that inhibition of CDK9 activity abolished the transcription of GV oocytes globally. Moreover, using fluorescence in situ hybridization, in absence of CDK9 kinase activity the production of ribosomal RNAs was impaired. We also presented the evidences indicating that P-TEFb kinase activity is essential for resumption of oocyte meiosis and embryo development. Treatment with CDK9 inhibitors resulted in germinal vesicle arrest in maturing oocytes in vitro. Inhibition of CDK9 kinase activity did not interfere with in vitro fertilization and pronuclear formation. However, when in vitro produced zygotes were treated with CDK9 inhibitors, their development beyond the 4-cell stage was impaired. In these embryos, inhibition of CDK9 abrogated global transcriptional activity and rRNA production. Collectively, our data suggested that P-TEFb kinase activity is crucial for oocyte maturation, embryo development and regulation of RNA transcription in pig.
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PMID:P-TEFb Kinase Activity Is Essential for Global Transcription, Resumption of Meiosis and Embryonic Genome Activation in Pig. 2701 Dec 7