Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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

By binding to the transactivation response element (TAR) RNA, the transcriptional transactivator (Tat) from the human immunodeficiency virus increases rates of elongation rather than initiation of viral transcription. Two cyclin-dependent serine/threonine kinases, CDK7 and CDK9, which phosphorylate the C-terminal domain of RNA polymerase II, have been implicated in Tat transactivation in vivo and in vitro. In this report, we demonstrate that CDK9, which is the kinase component of the positive transcription elongation factor b (P-TEFb) complex, can activate viral transcription when tethered to the heterologous Rev response element RNA via the regulator of expression of virion proteins (Rev). The kinase activity of CDK9 and cyclin T1 is essential for these effects. Moreover, P-TEFb binds to TAR only in the presence of Tat. We conclude that Tat-P-TEFb complexes bind to TAR, where CDK9 modifies RNA polymerase II for the efficient copying of the viral genome.
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PMID:The ability of positive transcription elongation factor B to transactivate human immunodeficiency virus transcription depends on a functional kinase domain, cyclin T1, and Tat. 969 9

The human immunodeficiency virus type 1 transcriptional regulator Tat increases the efficiency of elongation, and complexes containing the cellular kinase CDK9 have been implicated in this process. CDK9 is part of the Tat-associated kinase TAK and of the elongation factor P-TEFb (positive transcription elongation factor-b), which consists minimally of CDK9 and cyclin T. TAK and P-TEFb are both able to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II, but their relationships to one another and to the stimulation of elongation by Tat are not well characterized. Here we demonstrate that human cyclin T1 (but not cyclin T2) interacts with the activation domain of Tat and is a component of TAK as well as of P-TEFb. Rodent (mouse and Chinese hamster) cyclin T1 is defective in Tat binding and transactivation, but hamster CDK9 interacts with human cyclin T1 to give active TAK in hybrid cells containing human chromosome 12. Although TAK is phosphorylated on both serine and threonine residues, it specifically phosphorylates serine 5 in the CTD heptamer. TAK is found in the nuclear and cytoplasmic fractions of human cells as a large complex (approximately 950 kDa). Magnesium or zinc ions are required for the association of Tat with the kinase. We suggest a model in which Tat first interacts with P-TEFb to form the TAK complex that engages with TAR RNA and the elongating transcription complex, resulting in hyperphosphorylation of the CTD on serine 5 residues.
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PMID:Human and rodent transcription elongation factor P-TEFb: interactions with human immunodeficiency virus type 1 tat and carboxy-terminal domain substrate. 1036 92

The CDK9-cyclin T kinase complex, positive transcription elongation factor b (P-TEFb), stimulates the process of elongation of RNA polymerase (Pol) II during transcription of human immunodeficiency virus. P-TEFb associates with the human immunodeficiency virus Tat protein and with the transactivation response element to form a specific complex, thereby mediating efficient elongation. Here, we show that P-TEFb preferentially phosphorylates hSPT5 as compared with the carboxyl-terminal domain of RNA Pol II in vitro. Phosphorylation of hSPT5 by P-TEFb occurred on threonine and serine residues in its carboxyl-terminal repeat domains. In addition, we provide several lines of evidence that P-TEFb is a CDK-activating kinase (CAK)-independent kinase. For example, CDK9 was not phosphorylated by CAK, whereas CDK2-cyclin A kinase activity was dramatically enhanced by CAK. Therefore, it is likely that P-TEFb participates in regulation of elongation by RNA Pol II by phosphorylation of its substrates, hSPT5 and the CTD of RNA Pol II, in a CAK-independent manner.
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PMID:Positive transcription elongation factor B phosphorylates hSPT5 and RNA polymerase II carboxyl-terminal domain independently of cyclin-dependent kinase-activating kinase. 1114 67

Transcription and splicing are coordinated processes in mammalian cells. We have used affinity chromatography with immobilized transcription elongation factor SII to purify a protein complex that contains core RNA polymerase II (RNA Pol II), the general transcription initiation factors, and several splicing factors, including the U1, U2, and U4 small nuclear RNPs, the U2AF(65), and serine/arginine-rich proteins. The splicing factors and the transcription machinery co-purify through a gel filtration column and co-immunoprecipitate in experiments using an anti-U2AF(65) antibody, indicating that they are part of a unique complex. Although the RNA Pol II-containing complex does not possess splicing activity, it can complement small nuclear RNP-inactivated extracts and can promote the formation of a pre-spliceosome complex. Because interactions between components of the splicing and transcription machineries occur in the context of a complex containing a hypophosphorylated RNA Pol II capable of initiating transcription, our results suggest that the coupling between transcription and splicing begins before transcription initiation.
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PMID:A human RNA polymerase II-containing complex associated with factors necessary for spliceosome assembly. 1177 74

Since Candida rugosa utilizes a nonuniversal serine codon CUG rather than leucine, no vectors have been constructed to transform this organism. Moreover, it is difficult to design a new transformation system because no selection markers and promoters are available. In this study, Zeocin (400 microg/ml) was demonstrated to inhibit the growth of C. rugosa. The dominant selectable marker bleomycin-resistant determinant (ble) gene containing five CUG codons in an open-reading frame of 375 bp was synthesized by replacing its CUG codons into leucine codons (zeo-n). This marker conferred resistance to Zeocin. GAL1 promoter, transcription elongation factor 1 (TEF1) promoter from Saccharomyces cerevisiae and LIP3 promoter from C. rugosa were then used to drive zeo-n and to examine the function of promoter in C. rugosa. The resulting vectors enabled selection of Zeocin-resistant clones after transformation by LiCl method and electroporation. These results demonstrate that transformation into C. rugosa is feasible under the operation of GAL1, TEF1, and LIP3 promoters. The development of the transformation system for C. rugosa is essential to the genetic analysis of gene regulation and biochemical features of this fungal species and the expression of recombinant proteins in C. rugosa.
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PMID:A transformation system for the nonuniversal CUG(Ser) codon usage species Candida rugosa. 1245 43

The histone methyltransferase Set2, which specifically methylates lysine 36 of histone H3, has been shown to repress transcription upon tethering to a heterologous promoter. However, the mechanism of targeting and the consequence of Set2-dependent methylation have yet to be demonstrated. We sought to identify the protein components associated with Set2 to gain some insights into the in vivo function of this protein. Mass spectrometry analysis of the Set2 complex, purified using a tandem affinity method, revealed that RNA polymerase II (pol II) is associated with Set2. Immunoblotting and immunoprecipitation using antibodies against subunits of pol II confirmed that the phosphorylated form of pol II is indeed an integral part of the Set2 complex. Gst-Set2 preferentially binds to CTD synthetic peptides phosphorylated at serine 2, and to a lesser extent, serine 5 phosphorylated peptides, but has no affinity for unphosphorylated CTD, suggesting that Set2 associates with the elongating form of the pol II. Furthermore, we show that set2Delta ppr2Delta double mutants (PPR2 encodes TFIIS, a transcription elongation factor) are synthetically hypersensitive to 6-azauracil, and that deletions in the CTD reduce in vivo levels of H3 lysine 36 methylation. Collectively, these results suggest that Set2 is involved in regulating transcription elongation through its direct contact with pol II.
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PMID:The Set2 histone methyltransferase functions through the phosphorylated carboxyl-terminal domain of RNA polymerase II. 1251 61

A natural amino acid substitution in the human immunodeficiency virus type 1 (HIV-1) transcriptional activator Tat increases its activity and compensates for deleterious mutations elsewhere in the Tat protein. Substitution of asparagine for threonine 23 increases Tat transactivation of the HIV-1 promoter and the binding of Tat to the cellular kinase positive transcription elongation factor b (P-TEFb). Of nine other position 23 mutations tested, only the serine substitution retained wild-type activity. Correspondingly, asparagine is the most frequent amino acid at this position in HIV-1 isolates, followed by threonine and serine. Asparagine is prevalent in Tat proteins of viruses in clades A, C, and D, which are major etiologic agents of AIDS. We suggest that selection for asparagine in position 23 confers an advantage to the virus, since it can compensate for deleterious mutations in Tat. It may also support the replication of otherwise less fit drug-resistant viruses and permit the emergence of virulent strains.
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PMID:A naturally occurring substitution in human immunodeficiency virus Tat increases expression of the viral genome. 1285 33

Schizosaccharomyces pombe Cdk9/Pch1 protein kinase is a functional ortholog of the essential Saccharomyces cerevisiae Bur1/Bur2 kinase and a putative ortholog of metazoan P-TEFb (Cdk9/cyclin T). SpCdk9/Pch1 phosphorylates of the carboxyl-terminal domain (CTD) of the S. pombe transcription elongation factor Spt5, which consists of 18 tandem repeats of a nonapeptide of consensus sequence 1TPAWNSGSK9. We document the divalent cation dependence and specificity of SpCdk9/Pch1, its NTP dependence and specificity, the dependence of Spt5-CTD phosphorylation on the number of tandem nonamer repeats, and the specificity for phosphorylation of the Spt5-CTD on threonine at position 1 within the nonamer element. SpCdk9/Pch1 also phosphorylates the CTD heptaptide repeat array of the largest subunit of S. pombe RNA polymerase II (consensus sequence YSPTSPS) and does so exclusively on serine. SpCdk9/Pch1 catalyzes autophosphorylation of the kinase and cyclin subunits of the kinase complex. The distribution of phosphorylation sites on SpCdk9 (86% Ser(P), 11% Thr(P), 3% Tyr(P)) is distinct from that on Pch1 (2% Ser(P), 98% Thr(P)). We conducted a structure-guided mutational analysis of SpCdk9, whereby a total of 29 new mutations of 12 conserved residues were tested for in vivo function by complementation of a yeast bur1Delta mutant. We identified many lethal and conditional mutations of side chains implicated in binding ATP and the divalent cation cofactor, phosphoacceptor substrate recognition, and T-loop dynamics. We surmise that the lethality of the of T212A mutation in the T-loop reflects an essential phosphorylation event, insofar as the conservative T212S change rescued wild-type growth; the phosphomimetic T212E change rescued growth at 30 degrees C; and the effects of mutating the T-loop threonine were phenocopied by mutations in the three conserved arginines predicted to chelate the phosphate on the T-loop threonine.
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PMID:Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 protein kinase: Spt5 phosphorylation, autophosphorylation, and mutational analysis. 1290 90

The expression of the cytochrome P450 1A1 gene (cyp1a1) is regulated by the aryl hydrocarbon receptor (AhR), which is a ligand-activated transcription factor that mediates most toxic responses induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In the nucleus, ligand-activated AhR binds to the xenobiotic response elements, initiating chromatin remodeling and recruitment of coregulators, leading to the formation of preinitiation complex followed by elongation. Here, we report that ligand-activated AhR recruits the positive transcription elongation factor (P-TEFb) and RNA polymerase II (RNA PII) to the cyp1a1 promoter with concomitant phosphorylation of the RNA PII carboxyl domain (CTD). Interestingly, the serine 2 and serine 5 of the heptapeptide repeats (YSPTSPS) were sequentially phosphorylated upon TCDD treatment. Inhibition of P-TEFb kinase activity by 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole (DRB) suppressed CTD phosphorylation (especially serine 2 phosphorylation) and abolished processive elongation without disrupting the assembly of the preinitiation complex at the cyp1a1 promoter. Remarkably, we found that activation of NF-kappaB by TNF-alpha selectively inhibited TCDD-induced serine 2 phosphorylation in mouse liver cells, suggesting that residue-specific phosphorylation of RNA PII CTD at the cyp1a1 promoter is an important regulatory point upon which signal "cross-talk" converges. Finally, we show that ligand-activated AhR associated with P-TEFb through the C terminus of cyclin T1, suggesting that AhR recruit the P-TEFb to the cyp1a1 promoter whereupon its kinase subunit phosphorylates the RNA PII CTD.
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PMID:Interactions between the aryl hydrocarbon receptor and P-TEFb. Sequential recruitment of transcription factors and differential phosphorylation of C-terminal domain of RNA polymerase II at cyp1a1 promoter. 1291 20

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) promotes tumor progression through activation of matrix metalloproteinase (MMP) activity. MMP-9 is a gelatinase secreted by both cancer cells and surrounding stromal cells, and it contributes to TNF-alpha-stimulated tumor invasion and metastasis. Cyclin-dependent kinase 9 (CDK9), the catalytic component of positive transcription elongation factor-b, phosphorylates serine 2 residues in the C-terminal domain of RNA polymerase II for productive transcription elongation and is up-regulated upon exposure to various stresses. This study investigated roles of CDK9 in TNF-alpha-stimulated MMP-9 expression in human lung adenocarcinoma cells. CDK9 activity was inhibited using three different strategies, including the CDK9 pharmacological inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a dominant-negative CDK9, and a CDK9-specific small interfering RNA. All three approaches reduced TNF-alpha-mediated accumulation of MMP-9 in the conditioned media as demonstrated by gelatin zymography. In contrast, transforming growth factor-beta1-induced accumulation of MMP-2 was unaffected by DRB. Expression of the MMP-9 gene was examined using reverse transcription real time PCR and using a transient transfection assay to evaluate MMP-9 promoter activity. DRB reduced the TNF-alpha-induced increase in MMP-9 mRNA levels but did not effect transforming growth factor-beta1-induced MMP-2 mRNA expression. Consistently DRB and dominant-negative CDK9 completely abrogated TNF-alpha-stimulated human MMP-9 promoter activity. TNF-alpha did not regulate expression or localization of CDK9 or its regulatory partner Cyclin T. However, TNF-alpha stimulated CDK9 binding to Cyclin T and MMP-9 gene occupancy by both CDK9 and the serine 2-phosphorylated form of RNA polymerase II. Our findings indicate that CDK9 mediates TNF-alpha-induced MMP-9 transcription. Disruption of TNF-alpha signaling using CDK9 inhibitors could serve as a potential therapeutic strategy against tumor invasion and metastasis.
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PMID:Cyclin-dependent kinase 9 is required for tumor necrosis factor-alpha-stimulated matrix metalloproteinase-9 expression in human lung adenocarcinoma cells. 1552 90


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