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: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

HIV-1 gene expression and viral replication require the viral transactivator protein Tat. The RNA polymerase II transcriptional elongation factor P-TEFb (cyclin-dependent kinase 9/cyclin T) is a cellular protein kinase that has recently been shown to be a key component of the Tat-transactivation process. For this report, we studied the requirement for P-TEFb in HIV-1 infection, and we now show that P-TEFb is both essential and limiting for HIV-1 replication. Attenuation of P-TEFb kinase activity either by expression of a dominant-negative cyclin-dependent kinase 9 transgene or through the use of small-molecule inhibitors suppresses HIV-1 gene expression and HIV-1 replication. Inhibition of HIV-1 replication is affected in a manner consistent with a direct and specific effect on P-TEFb and the known functional role of P-TEFb in Tat-activated transcription. Tat-activated expression of HIV-1 genes seems uniquely dependent on P-TEFb, as inhibition of P-TEFb activity and HIV-1 replication can be achieved without compromising cell viability or RNA polymerase II-dependent cellular gene transcription. Selective inhibition of the P-TEFb kinase may therefore provide a novel approach for developing chemotherapeutic agents against HIV-1.
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PMID:Host-cell positive transcription elongation factor b kinase activity is essential and limiting for HIV type 1 replication. 1037 93

Recent studies have demonstrated roles for Spt4, Spt5, and Spt6 in the regulation of transcriptional elongation in both yeast and humans. Here, we show that Drosophila Spt5 and Spt6 colocalize at a large number of transcriptionally active chromosomal sites on polytene chromosomes and are rapidly recruited to endogenous and transgenic heat shock loci upon heat shock. Costaining with antibodies to Spt6 and to either the largest subunit of RNA polymerase II or cyclin T, a subunit of the elongation factor P-TEFb, reveals that all three factors have a similar distribution at sites of active transcription. Crosslinking and immunoprecipitation experiments show that Spt5 is present at uninduced heat shock gene promoters, and that upon heat shock, Spt5 and Spt6 associate with the 5' and 3' ends of heat shock genes. Spt6 is recruited within 2 minutes of a heat shock, similar to heat shock factor (HSF); moreover, this recruitment is dependent on HSF. These findings provide support for the roles of Spt5 in promoter-associated pausing and of Spt5 and Spt6 in transcriptional elongation in vivo.
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PMID:High-resolution localization of Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and transcription elongation. 1104 Feb 17

Regulation of HIV-1 gene expression by the viral Tat transactivator is a critical step in the viral life cycle. Tat acts as a highly unusual transcription factor that interacts with a stem-loop RNA structure (TAR) found at the 5' end of all viral transcripts. There, it induces a modification of chromatin at the HIV-1 long terminal repeat (LTR) promoter and stimulates the recruitment of elongation-competent RNA polymerase II complexes capable of processive transcription. Increase of transcriptional elongation is the consequence of the interaction of Tat with cyclin T1, the cyclin component of CDK9, which phosphorylates the carboxy-terminal domain of RNA polymerase II to enhance its processivity. Tat-induced transcriptional activation of the LTR promoter is concomitant with recruitment of the transcriptional coactivators p300 and the highly homologue cAMP-responsive transcription factor binding protein (CBP). These large proteins act at the level of transcriptional initiation by bridging the basal transcription machinery with specific transcriptional activators. Furthermore, p300/CBP are histone acetyl-transferases capable of modulating the interaction of nucleosomes with DNA and with chromatin remodeling complexes. Besides histones, Tat itself is a substrate for the enzymatic activity of p300/CBP and of the associated factor P/CAF, suggesting a regulatory role of acetylation on the protein itself. Devising a unifying model for LTR activation that includes activities of Tat at the levels of both transcriptional initiation and transcriptional elongation is a challenging task at this moment. Nevertheless, protein localization studies indicate that both cyclin T1 and p300/CBP co-localize in specific subnuclear compartments, thus suggesting participation of both proteins in the formation of multimolecular complexes governing coordinated steps of transcriptional activation.
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PMID:Multiple modes of transcriptional regulation by the HIV-1 Tat transactivator. 1154 19

Transcription and pre-mRNA splicing are tightly coupled gene expression events in eukaryotic cells. An interaction between the carboxy-terminal domain of the largest subunit of RNA polymerase (Pol) II and components of the splicing machinery is postulated to mediate this coupling. Here, we show that splicing factors function directly to promote transcriptional elongation, demonstrating that transcription is more intimately coupled to splicing than previously thought. The spliceosomal U small nuclear ribonucleoproteins (snRNPs) interact with human transcription elongation factor TAT-SF1 (refs 6,7,8,9) and strongly stimulate polymerase elongation when directed to an intron-free human immunodeficiency virus-1 (HIV-1) template. This effect is likely to be mediated through the binding of TAT-SF1 to elongation factor P-TEFb, a proposed component of the transcription elongation complex. Inclusion of splicing signals in the nascent transcript further stimulates transcription, supporting the notion that the recruitment of U snRNPs near the elongating polymerase is important for transcription. Because the TAT-SF1-U snRNP complex also stimulates splicing in vitro, it may serve as a dual-function factor to couple transcription and splicing and to facilitate their reciprocal activation.
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PMID:Stimulatory effect of splicing factors on transcriptional elongation. 1178 68

RNA polymerase II (pol II) is subject to an early elongation delay induced by negative factors Spt5/Spt4 and NELF, which is overcome by the positive factor P-TEFb (Cdk9/cyclin T), a protein kinase that phosphorylates the pol II C-terminal domain (CTD) and the transcription elongation factor Spt5. Although the rationale for this arrest and restart is unclear, recent studies suggest a connection to mRNA capping, which is coupled to transcription elongation via physical and functional interactions between the cap-forming enzymes, the CTD-PO(4), and Spt5. Here we identify a novel interaction between fission yeast RNA triphosphatase Pct1, the enzyme that initiates cap formation, and Schizosaccharomyces pombe Cdk9. The C-terminal segment of SpCdk9 comprises a Pct1-binding domain distinct from the N-terminal Cdk domain. We show that the Cdk domain interacts with S. pombe Pch1, a homolog of cyclin T, and that the purified recombinant SpCdk9/Pch1 heterodimer can phosphorylate both the pol II CTD and the C-terminal domain of S. pombe Spt5. We provide genetic evidence that SpCdk9 and Pch1 are functional orthologs of the Saccharomyces cerevisiae CTD kinase Bur1/Bur2, a putative yeast P-TEFb. Mutations of the kinase active site and the regulatory T-loop of SpCdk9 abolish its activity in vivo. Deleting the C-terminal domain of SpCdk9 causes a severe growth defect. We suggest a model whereby Spt5-induced arrest of early elongation ensures a temporal window for recruitment of the capping enzymes, which in turn attract Cdk9 to alleviate the arrest. This elongation checkpoint may avoid wasteful rounds of transcription of uncapped pre-mRNAs.
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PMID:Interactions between fission yeast Cdk9, its cyclin partner Pch1, and mRNA capping enzyme Pct1 suggest an elongation checkpoint for mRNA quality control. 1247 73

The tumor suppressor gene product BRCA1 is a component of the RNA polymerase II (pol II) holoenzyme that is involved, through binding to various regulatory proteins, in either activation or repression of transcription. Using a yeast two-hybrid screen, we have identified a human zinc-finger-containing protein NUFIP that interacts with BRCA1. The ubiquitous, stably expressed, nuclear protein NUFIP specifically stimulates activator-independent pol II transcription in vitro and in vivo. Immunodepletion of the endogenous NUFIP causes a marked decrease of pol II transcription, which is then shown to be restored by stable complex of ectopically produced NUFIP and associated factors. NUFIP not only interacts with BRCA1 but also associates with the positive elongation factor P-TEFb through interaction with the regulatory Cyclin T1 subunit. Cyclin T1 is required for BRCA1- and NUFIP-dependent synergistic activation of pol II transcription in 293 cells. Mutation of the zinc-finger domain abolishes the NUFIP-mediated transcriptional activation. We show that NUFIP is associated with preinitiation complexes, open transcription complexes, and elongation complexes. In addition, NUFIP facilitates ATP-dependent dissociation of hyperphosphorylated pol II from open transcription complexes in vitro.
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PMID:BRCA1 cooperates with NUFIP and P-TEFb to activate transcription by RNA polymerase II. 1510 25

STAT transcription factors (signal transducers and activators of transcription) are cytoplasmic proteins that induce gene activation in response to cytokine receptor stimulation. Following tyrosine phosphorylation, STAT proteins translocate into the nucleus and activate specific target genes. We have previously reported that STAT3 activates the expression of the p21waf1 gene through its association with the NcoA/SRC1a and CBP coactivators. In this study, we explore the role of BRG1, a component of the SWI/SNF chromatin-remodeling complex, and the role of cdk9, a component of the elongation factor P-TEFb, in the STAT3-mediated expression of p21waf1. We found using pull-down experiments and co-immunoprecipitation assays that both proteins associate with STAT3. Chromatin immunoprecipitation (ChIP) experiments indicate that STAT3 DNA binding results in histone H3 acetylation and BRG1 recruitment. Using Southern blot analysis, we found that the loading of BRG1 is followed by an increased accessibility of the proximal p21waf1 promoter and by the association of RNA polymerase II. As a next step, STAT3 then recruits the cdk9 kinase to phosphorylate the carboxy-terminal domain of the RNA polymerase at serine 2. Accordingly, the elongating form of the polymerase can be detected by ChIP experiments on the coding region of the gene, probably initiating mRNA synthesis. Therefore, STAT3 not only promotes the initiation of transcription but also regulates chromatin remodeling and transcription elongation through its interaction with BRG1 and cdk9.
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PMID:Implication of BRG1 and cdk9 in the STAT3-mediated activation of the p21waf1 gene. 1528 5

Formation of productive transcription complexes after promoter escape by RNA polymerase II is a major event in eukaryotic gene regulation. Both negative and positive factors control this step. The principal negative elongation factor (NELF) contains four polypeptides and requires for activity the two-polypeptide 5,6-dichloro-1-beta-D-ribobenzimidazole-sensitivity inducing factor (DSIF). DSIF/NELF inhibits early transcript elongation until it is counteracted by the positive elongation factor P-TEFb. We report a previously undescribed activity of DSIF/NELF, namely inhibition of the transcript cleavage factor TFIIS. These two activities of DSIF/NELF appear to be mechanistically distinct. Inhibition of nucleotide addition requires > or = 18 nt of nascent RNA, whereas inhibition of TFIIS occurs at all transcript lengths. Because TFIIS promotes escape from promoter-proximal pauses by stimulating cleavage of back-tracked nascent RNA, TFIIS inhibition may help DSIF/NELF negatively regulate productive transcription.
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PMID:A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS. 1621 96

Eukaryotic organisms possess a host of factors that regulate transcriptional elongation. In higher eukaryotes, the transcription factor P-TEFb not only regulates phosphorylation of the RNA polymerase II C-terminal domain, but it also inhibits the action of transcriptional repressors and is required for the association of several elongation factors with the transcribing polymerase. In the yeast Saccharomyces cerevisiae, the cyclin dependent kinases Bur1/Bur2 and Ctk complex (Ctk1, 2 and 3) are also able to impact several aspects of transcription. Together, these two kinase complexes appear to functionally reconstitute the activity of P-TEFb in yeast. Recent findings regarding the role of these kinases in histone tail modifications and transcriptional regulation is briefly reviewed below.
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PMID:Bur1/Bur2 and the Ctk complex in yeast: the split personality of mammalian P-TEFb. 1672 Oct 54


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