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 positive transcription elongation factor, P-TEFb, controls the fraction of initiated RNA polymerase II molecules that enter into the productive mode of elongation necessary to generate mRNAs. To better understand the mechanism of this transition into productive elongation we optimized a defined in vitro transcription system and compared results obtained with it to those obtained with a crude system. We found that controlling the function of TFIIF is a key aspect of RNA polymerase II elongation control. Before P-TEFb function, early elongation complexes under the control of negative factors are completely unresponsive to the robust elongation stimulatory activity of TFIIF. P-TEFb-mediated phosphorylation events, targeting the elongation complex containing DSIF and NELF, reverse the negative effect of DSIF and NELF and simultaneously facilitate the action of TFIIF. We also found that productive elongation complexes are completely resistant to negative elongation factors. Our data suggest that an additional factor(s) is involved in establishing the unique resistance activities of the elongation complexes before and after P-TEFb function. Furthermore, we provide evidence for the existence of another positive activity required for efficient function of P-TEFb. A model of the mechanism of P-TEFb-mediated elongation control is proposed in which P-TEFb induces the transition into productive elongation by changing the accessibility of elongation factors to elongation complexes. Our results have uncovered important properties of elongation complexes that allow a more complete understanding of how P-TEFb controls the elongation phases of transcription by RNA polymerase II.
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PMID:Properties of RNA polymerase II elongation complexes before and after the P-TEFb-mediated transition into productive elongation. 1754 48

The positive elongation factor P-TEFb appears to function as a crucial C-terminal-domain (CTD) kinase for RNA polymerase II (Pol II) transcribing immediate early genes (IEGs) in neuroendocrine GH4C1 cells. Chromatin immunoprecipitation indicated that in resting cells Pol II occupied the promoter-proximal regions of the c-fos and junB genes, together with the negative elongation factors DSIF and NELF. Thyrotropin-releasing hormone (TRH)-induced recruitment of positive transcription elongation factor b (P-TEFb) abolished the pausing of Pol II and enhanced phosphorylation of CTD serine 2, resulting in transcription elongation. In addition, P-TEFb was essential for splicing and 3'-end processing of IEG transcripts. Importantly, the MEK1-extracellular signal-regulated kinase (ERK) signaling pathway activated by TRH up-regulated nuclear CDK9 and CDK9/cyclinT1 dimers (i.e., P-TEFb), facilitating the recruitment of P-TEFb to c-fos and other IEGs. Thus, in addition to established gene transcription control via promoter response elements, the MEK1-ERK signaling pathway controls transcription elongation by Pol II via the up-regulation of nuclear CDK9 integrated into P-TEFb.
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PMID:Up-regulation of P-TEFb by the MEK1-extracellular signal-regulated kinase signaling pathway contributes to stimulated transcription elongation of immediate early genes in neuroendocrine cells. 1808 94

Efficient HIV-1 transcription requires the induction of cellular transcription factors, such as NF-kappaB, and the viral factor Tat, which through the recruitment of P-TEFb enhances processive transcription. However, whether cellular signals repress HIV-1 transcription to establish proviral latency has not been well studied. Previously, it has been shown that the receptor tyrosine kinase RON inhibits HIV transcription. To gain insights into the biochemical mechanisms by which RON inhibits transcription we examined the binding of transcription factors to the HIV provirus long terminal repeat using chromatin immunoprecipitation. RON expression decreased basal levels of NF-kappaB and RNA polymerase II (Pol II) binding to the HIV provirus long terminal repeat but did not prevent the induction of these complexes following treatment with cytokines. However, RON did decrease efficient transcription elongation because reduced RNA Pol II was associated with HIV-1 genomic sequences downstream of the transcriptional start site. There was a correlation between RON expression and increased binding of factors that negatively regulate transcription elongation, NELF, Spt5, and Pcf11. Furthermore, the ability of RON to inhibit HIV-1 transcription was sensitive to a histone deacetylase inhibitor and was associated with nucleosome remodeling. These results indicate that RON represses HIV transcription at multiple transcriptional check points including initiation, elongation and chromatin organization and are the first studies to show that cellular signaling pathways target Pol II pausing to repress gene expression.
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PMID:The receptor tyrosine kinase RON represses HIV-1 transcription by targeting RNA polymerase II processivity. 1820 63

Recent analyses of RNA polymerase II (Pol II) revealed that Pol II is concentrated at the promoters of many active and inactive genes. NELF causes Pol II to pause in the promoter-proximal region of the hsp70 gene in Drosophila melanogaster. In this study, genome-wide location analysis (chromatin immunoprecipitation-microarray chip [ChIP-chip] analysis) revealed that NELF is concentrated at the 5' ends of 2,111 genes in Drosophila cells. Permanganate genomic footprinting was used to determine if paused Pol II colocalized with NELF. Forty-six of 56 genes with NELF were found to have paused Pol II. Pol II pauses 30 to 50 nucleotides downstream from transcription start sites. Analysis of DNA sequences in the vicinity of paused Pol II identified a conserved DNA sequence that probably associates with TFIID but detected no evidence of RNA secondary structures or other conserved sequences that might directly control elongation. ChIP-chip experiments indicate that GAGA factor associates with 39% of the genes that have NELF. Surprisingly, NELF associates with almost one-half of the most highly expressed genes, indicating that NELF is not necessarily a repressor of gene expression. NELF-associated pausing of Pol II might be an obligatory but sometimes transient checkpoint during the transcription cycle.
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PMID:NELF and GAGA factor are linked to promoter-proximal pausing at many genes in Drosophila. 1833 13

The eukaryotic transcription elongation factor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) sensitivity inducing factor (DSIF), is involved in regulating the processivity of RNA polymerase II. DSIF plays also a role in transcriptional activation, and in concert with the negative elongation factor NELF causes promoter proximal pausing of RNA polymerase II. Furthermore, DSIF has also been implicated in regulating the transcription of the human immunodeficiency virus proviral DNA. Human DSIF is composed of the two subunits, hSpt4 (p14) and hSpt5 (p160), corresponding to the yeast homologs Spt4 and Spt5. Here we show the purification and characterization of the small subunit, hSpt4. We were able to purify the protein in a soluble form separately from the larger hSpt5 subunit. CD and NMR spectroscopy show that the purified protein hSpt4 exhibits an alpha/beta topology with a well defined tertiary structure. Furthermore metal analysis by ICP-OES indicates that the protein contains a functional 4-Cys Zn-finger.
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PMID:The small hSpt4 subunit of the human transcription elongation factor DSIF is a Zn-finger protein with alpha/beta type topology. 1837 78

Insulator elements play a role in gene regulation that is potentially linked to nuclear organization. Boundary element-associated factors (BEAFs) 32A and 32B associate with hundreds of sites on Drosophila polytene chromosomes. We hybridized DNA isolated by chromatin immunoprecipitation to genome tiling microarrays to construct a genome-wide map of BEAF binding locations. A distinct difference in the association of 32A and 32B with chromatin was noted. We identified 1,820 BEAF peaks and found that more than 85% were less than 300 bp from transcription start sites. Half are between head-to-head gene pairs. BEAF-associated genes are transcriptionally active as judged by the presence of RNA polymerase II, dimethylated histone H3 K4, and the alternative histone H3.3. Forty percent of these genes are also associated with the polymerase negative elongation factor NELF. Like NELF-associated genes, most BEAF-associated genes are highly expressed. Using quantitative reverse transcription-PCR, we found that the expression levels of most BEAF-associated genes decrease in embryos and cultured cells lacking BEAF. These results provide an unexpected link between BEAF and transcription, suggesting that BEAF plays a role in maintaining most associated promoter regions in an environment that facilitates high transcription levels.
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PMID:Genome-wide mapping of boundary element-associated factor (BEAF) binding sites in Drosophila melanogaster links BEAF to transcription. 1938 Apr 83

The negative elongation factor NELF is a key component of an early elongation checkpoint generally located within 100 bp of the transcription start site of protein-coding genes. Negotiation of this checkpoint and conversion to productive elongation require phosphorylation of the carboxy-terminal domain of RNA polymerase II (pol II), NELF, and DRB sensitivity-inducing factor (DSIF) by positive transcription elongation factor b (P-TEFb). P-TEFb is dispensable for transcription of the noncoding U2 snRNA genes, suggesting that a NELF-dependent checkpoint is absent. However, we find that NELF at the end of the 800-bp U2 gene transcription unit and RNA interference-mediated knockdown of NELF causes a termination defect. NELF is also associated 800 bp downstream of the transcription start site of the beta-actin gene, where a "late" P-TEFb-dependent checkpoint occurs. Interestingly, both genes have an extended nucleosome-depleted region up to the NELF-dependent control point. In both cases, transcription through this region is P-TEFb independent, implicating chromatin in the formation of the terminator/checkpoint. Furthermore, CTCF colocalizes with NELF on the U2 and beta-actin genes, raising the possibility that it helps the positioning and/or function of the NELF-dependent control point on these genes.
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PMID:Chromatin structure is implicated in "late" elongation checkpoints on the U2 snRNA and beta-actin genes. 1945 Dec 31

The function of human TFIIH-associated Cdk7 in RNA polymerase II (Pol II) transcription and C-terminal domain (CTD) phosphorylation was investigated in analogue-sensitive Cdk7(as/as) mutant cells where the kinase can be inhibited without disrupting TFIIH. We show that both Cdk7 and Cdk9/PTEFb contribute to phosphorylation of Pol II CTD Ser5 residues on transcribed genes. Cdk7 is also a major kinase of CTD Ser7 on Pol II at the c-fos and U snRNA genes. Furthermore, TFIIH and recombinant Cdk7-CycH-Mat1 as well as recombinant Cdk9-CycT1 phosphorylated CTD Ser7 and Ser5 residues in vitro. Inhibition of Cdk7 in vivo suppressed the amount of Pol II accumulated at 5' ends on several genes including c-myc, p21, and glyceraldehyde-3-phosphate dehydrogenase genes, indicating reduced promoter-proximal pausing or polymerase "leaking" into the gene. Consistent with a 5' pausing defect, Cdk7 inhibition reduced recruitment of the negative elongation factor NELF at start sites. A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36-two marks of elongation-within genes when the kinase was inhibited. Consistent with a new role for TFIIH at 3' ends, it was detected within genes and 3'-flanking regions, and Cdk7 inhibition delayed pausing and transcription termination.
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PMID:TFIIH-associated Cdk7 kinase functions in phosphorylation of C-terminal domain Ser7 residues, promoter-proximal pausing, and termination by RNA polymerase II. 1966 75

The eukaryotic transcription elongation factor DSIF [DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) sensitivity-inducing factor] is composed of two subunits, hSpt4 and hSpt5, which are homologous to the yeast factors Spt4 and Spt5. DSIF is involved in regulating the processivity of RNA polymerase II and plays an essential role in transcriptional activation of eukaryotes. At several eukaryotic promoters, DSIF, together with NELF (negative elongation factor), leads to promoter-proximal pausing of RNA polymerase II. In the present paper we describe the crystal structure of hSpt4 in complex with the dimerization region of hSpt5 (amino acids 176-273) at a resolution of 1.55 A (1 A=0.1 nm). The heterodimer shows high structural similarity to its homologue from Saccharomyces cerevisiae. Furthermore, hSpt5-NGN is structurally similar to the NTD (N-terminal domain) of the bacterial transcription factor NusG. A homologue for hSpt4 has not yet been found in bacteria. However, the archaeal transcription factor RpoE" appears to be distantly related. Although a comparison of the NusG-NTD of Escherichia coli with hSpt5 revealed a similarity of the three-dimensional structures, interaction of E. coli NusG-NTD with hSpt4 could not be observed by NMR titration experiments. A conserved glutamate residue, which was shown to be crucial for dimerization in yeast, is also involved in the human heterodimer, but is substituted for a glutamine residue in Escherichia coli NusG. However, exchanging the glutamine for glutamate proved not to be sufficient to induce hSpt4 binding.
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PMID:Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface. 1986 Jul 41

Transcription elongation factor DSIF/Spt4-Spt5 is capable of promoting and inhibiting RNA polymerase II elongation and is involved in the expression of various genes. While it has been known for many years that DSIF inhibits elongation in collaboration with the negative elongation factor NELF, how DSIF promotes elongation is largely unknown. Here, an activity-based biochemical approach was taken to understand the mechanism of elongation activation by DSIF. We show that the Paf1 complex (Paf1C) and Tat-SF1, two factors implicated previously in elongation control, collaborate with DSIF to facilitate efficient elongation. In human cells, these factors are recruited to the FOS gene in a temporally coordinated manner and contribute to its high-level expression. We also show that elongation activation by these factors depends on P-TEFb-mediated phosphorylation of the Spt5 C-terminal region. A clear conclusion emerging from this study is that a set of elongation factors plays nonredundant, cooperative roles in elongation. This study also shows unambiguously that Paf1C, which is generally thought to have chromatin-related functions, is involve directlyd in elongation control.
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PMID:DSIF, the Paf1 complex, and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II elongation. 1995 11


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