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 translation of mRNA in eukaryotic cells is regulated by amino acids through multiple mechanisms. One such mechanism involves activation of mTOR (Fig. 1). mTOR controls a myriad of downstream effectors, including RNA polymerase I, S6K1, 4E-BP1, and eEF2 kinase. In yeast, and probably in higher eukaryotes, mTOR signals through Tap42p/alpha 4 to regulate protein phosphatases. Through phosphorylation of Tap42p/alpha 4, mTOR abrogates dephosphorylation of the downstream effectors by PP2 A and/or PP6, resulting in their increased phosphorylation. Although at this time still speculative, in vitro results using mTOR immunoprecipitates suggest that mTOR, or an associated kinase, may also be directly involved in phosphorylating some effectors. Enhanced RNA polymerase I activity results in increased transcription of rDNA genes, whereas increased S6K1 activity promotes preferential translation of TOP mRNAs, such as those encoding ribosomal proteins. Together, stimulated RNA polymerase I and S6K1 activities enhance ribosome biogenesis, increasing the translational capacity of the cell. Phosphorylation of 4E-BP1 prohibits its association with eIF4E, allowing eIF4E to bind to eIF4G and form the active eIF4F complex. Increased eIF4F formation preferentially stimulates translation of mRNAs containing long, highly-structured 5' UTRs. Finally, amino acids cause inhibition of the eEF2 kinase, resulting in an increase in the proportion of eEF2 in the active, dephosphorylated form. By inhibiting eEF2 phosphorylation, amino acids may not only stimulate translation elongation, but may also prevent activation of GCN2 by enhancing the rate of removal of deacylated tRNA from the P-site on the ribosome; a potential activator of GCN2. GCN2 may also be regulated directly by the accumulation of deacylated-tRNA caused by treatment with inhibitors of tRNA synthetases or in cells incubated in the absence of essential amino acids. However, because the Km of the tRNA synthetases for amino acids is well above the amino acid concentrations found in plasma of fasted animals, such a mechanism may not be operative in mammals in vivo. Activation of GCN2 results in increased phosphorylation of the alpha-subunit of eIF2, which in turn causes inhibition of eIF2B. Thus, by preventing activation of GCN2, amino acids preserve eIF2B activity, which promotes translation of all mRNAs, i.e., global protein synthesis is enhanced.
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PMID:Regulation of translation initiation by amino acids in eukaryotic cells. 1157 65

During mitosis, the cyclin-dependent kinase, Cdc2, signals the inactivation of major anabolic processes such as transcription, mRNA processing, translation, and ribosome biogenesis, thereby providing energy needed for the radical and energetically costly structural reorganization of the cell. This is accomplished by phosphorylation and inactivation of several key anabolic elements, including TFIIIB, TFIID, RNA polymerase II, poly(A) polymerase, and translation elongation factor 1gamma. We report here that ribosomal S6 kinase 1 (S6K1), a protein kinase linked to the translation of ribosomal protein mRNAs, is also subject to regulation by Cdc2 in mitosis. In mitotic HeLa cells, when the activity of Cdc2 is high, S6K1 is phosphorylated at multiple Ser/Thr, Pro (S/TP) sites, including Ser(371), Ser(411), Thr(421), and Ser(424). Concomitant with this, the phosphorylation of the hydrophobic motif site, Thr(389), is reduced resulting in a decrease in the specific activity of S6K1. The mitotic S/TP phosphorylation sites are readily phosphorylated by Cdc2.cyclin B in vitro. These proline-directed phosphorylations are sensitive to chemical inhibitors of Cdc2 but not to inhibitors of mammalian target of rapamycin, phosphatidylinositol 3-kinase, MEK1/2, or p38. In murine FT210 cells arrested in mitosis, conditional inactivation of Cdc2 reduces phosphorylation of S6K1 at S/TP sites while simultaneously increasing phosphorylation of Thr(389) and of the S6K1 substrate, RPS6. A physical interaction exists between Cdc2 and S6K1, and this interaction is enhanced in mitotic cells. These results suggest that Cdc2 provides a signal that triggers inactivation of S6K1 in mitosis, presumably serving to spare energy for costly mitotic processes at the expense of ribosomal protein synthesis.
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PMID:Mitotic regulation of ribosomal S6 kinase 1 involves Ser/Thr, Pro phosphorylation of consensus and non-consensus sites by Cdc2. 1258 35

Regulation of ribosomal RNA gene transcription by RNA polymerase I (Pol I) is fundamental to ribosome biogenesis and therefore protein translation capacity and cell growth, yet little is known of the key signaling cascades involved. We show here that insulin-like growth factor-1 (IGF-1)-induced Pol I transcription in HEK293 cells is entirely dependent on phosphatidylinositol 3-kinase (PI3K) activity and, additionally, is modulated by the mammalian target of rapamycin (mTOR), which coordinates Pol I transcription with the availability of amino acids. The mitogen-activated protein kinase (MAPK) pathway is weakly stimulated by IGF-1 in these cells and partly contributes to Pol I transcription regulation. Activation of Pol I transcription by IGF-1 results from enhancement of the activity of the Pol I transcription machinery and increased occupancy by SL1 of the endogenous tandemly repeated ribosomal promoters in vivo. The inputs from PI3K, mTOR, and MAPK pathways converge to direct appropriate rRNA gene expression by Pol I in the nucleolus of mammalian cells in response to environmental cues, such as growth factors and nutrients.
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PMID:Phosphatidylinositol 3-kinase and mTOR signaling pathways regulate RNA polymerase I transcription in response to IGF-1 and nutrients. 3241 13

In cycling cells, transcription of ribosomal RNA genes by RNA polymerase I (Pol I) is tightly coordinated with cell growth. Here, we show that the mammalian target of rapamycin (mTOR) regulates Pol I transcription by modulating the activity of TIF-IA, a regulatory factor that senses nutrient and growth-factor availability. Inhibition of mTOR signaling by rapamycin inactivates TIF-IA and impairs transcription-initiation complex formation. Moreover, rapamycin treatment leads to translocation of TIF-IA into the cytoplasm. Rapamycin-mediated inactivation of TIF-IA is caused by hypophosphorylation of Se 44 (S44) and hyperphosphorylation of Se 199 (S199). Phosphorylation at these sites affects TIF-IA activity in opposite ways, for example, phosphorylation of S44 activates and S199 inactivates TIF-IA. The results identify a new target formTOR-signaling pathways and elucidate the molecular mechanism underlying mTOR-dependent regulation of RNA synthesis.
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PMID:mTOR-dependent activation of the transcription factor TIF-IA links rRNA synthesis to nutrient availability. 1500 9

PTEN is a tumor suppressor whose function is frequently lost in human cancer. It possesses a lipid phosphatase activity that represses the activation of PI3 kinase/Akt signaling, leading to decreased cell growth, proliferation, and survival. The potential for PTEN to regulate transcription of the large rRNAs by RNA polymerase I (RNA Pol I) was investigated. As increased synthesis of rRNAs is a hallmark of neoplastic transformation, the ability of PTEN to control the transcription of rRNAs might be crucial for its tumor suppressor function. The expression of PTEN in PTEN-deficient cells represses RNA Pol I transcription, while decreasing PTEN expression enhances transcription. PTEN-mediated repression requires its lipid phosphatase activity and is independent of the p53 status of the cell. This event can be uncoupled from PTEN's ability to regulate the cell cycle. RNA Pol I is regulated through PI3 kinase/Akt/mammalian target of rapamycin/S6 kinase, and the expression of constitutively activated S6 kinase is able to abrogate transcription repression by PTEN. No change in the expression of the RNA Pol I transcription components, upstream binding factor or SL1, was observed upon PTEN expression. However, chromatin immunoprecipitation assays demonstrate that PTEN differentially reduces the occupancy of the SL1 subunits on the rRNA gene promoter. Furthermore, PTEN induces dissociation of the SL1 subunits. Together, these results demonstrate that PTEN represses RNA Pol I transcription through a novel mechanism that involves disruption of the SL1 complex.
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PMID:PTEN represses RNA Polymerase I transcription by disrupting the SL1 complex. 1605 4

PTEN, a tumor suppressor whose function is frequently lost in human cancers, possesses a lipid phosphatase activity that represses phosphatidylinositol 3-kinase (PI3K) signaling, controlling cell growth, proliferation, and survival. The potential for PTEN to regulate the synthesis of RNA polymerase (Pol) III transcription products, including tRNAs and 5S rRNAs, was evaluated. The expression of PTEN in PTEN-deficient cells repressed RNA Pol III transcription, whereas decreased PTEN expression enhanced transcription. Transcription repression by PTEN was uncoupled from PTEN-mediated effects on the cell cycle and was independent of p53. PTEN acts through its lipid phosphatase activity, inhibiting the PI3K/Akt/mTOR/S6K pathway to decrease transcription. PTEN, through the inactivation of mTOR, targets the TFIIIB complex, disrupting the association between TATA-binding protein and Brf1. Kinetic analysis revealed that PTEN initially induces a decrease in the serine phosphorylation of Brf1, leading to a selective reduction in the occupancy of all TFIIIB subunits on tRNA(Leu) genes, whereas prolonged PTEN expression results in the enhanced serine phosphorylation of Bdp1. Together, these results demonstrate a new class of genes regulated by PTEN through its ability to repress the activation of PI3K/Akt/mTOR/S6K signaling.
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PMID:PTEN represses RNA polymerase III-dependent transcription by targeting the TFIIIB complex. 1839 Oct 23

The aim of this study was to develop an efficient cell-free protein expression system derived from mammalian cells. We established a HeLa cell-based in vitro coupled transcription/translation system with T7 RNA polymerase and a plasmid that harbored a T7 promoter/terminator unit. To enhance protein synthesis in the coupled system, we placed the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or the hepatitis C virus (HCV) IRES between the T7 promoter and the coding region of the plasmid. Remarkably, we found that these IRES-dependent systems were able to produce large proteins including GCN2 (160 kD), Dicer (200 kD) and mTOR (260 kD) to levels detectable on SDS-PAGE by Comassie Brilliant Blue-staining. We purified the synthesized proteins to near homogeneity, and validated their functionalities in the appropriate biochemical assays. In conclusion, the HeLa cell-based in vitro coupled transcription/translation system using the EMCV or HCV IRES is a convenient tool, particularly for the production of large recombinant proteins.
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PMID:A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins. 1881 49

The endoplasmic reticulum (ER) is the major cellular compartment where folding and maturation of secretory and membrane proteins take place. When protein folding needs exceed the capacity of the ER, the unfolded protein response (UPR) pathway modulates gene expression and downregulates protein translation to restore homeostasis. Here, we report that the UPR downregulates the synthesis of rRNA by inactivation of the RNA polymerase I basal transcription factor RRN3/TIF-IA. Inhibition of rRNA synthesis does not appear to involve the well-characterized mTOR (mammalian target of rapamycin) pathway; instead, PERK-dependent phosphorylation of eIF2alpha plays a critical role in the inactivation of RRN3/TIF-IA. Downregulation of rRNA transcription occurs simultaneously or slightly prior to eIF2alpha phosphorylation-induced translation repression. Since rRNA is the most abundant RNA species, constituting approximately 90% of total cellular RNA, its downregulation exerts a significant impact on cell physiology. Our study demonstrates the first link between regulation of translation and rRNA synthesis with phosphorylation of eIF2alpha, suggesting that this pathway may be broadly utilized by stresses that activate eIF2alpha kinases in order to coordinately regulate translation and ribosome biogenesis during cellular stress.
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PMID:Phosphorylation of eukaryotic translation initiation factor 2alpha coordinates rRNA transcription and translation inhibition during endoplasmic reticulum stress. 1947 Jul 60

Target of rapamycin (TOR) is a conserved regulator of gene expression from yeast to humans. In budding yeast, TOR is associated with ribosomal DNA (rDNA) promoter, which is critical for ribosome biogenesis and transfer RNA (tRNA) synthesis. Whether mTOR behaves similarly in mammalian cells is unknown. Here, we report that mTOR is detected at several different promoters in human and murine cells, including that of rDNA and tRNA genes. The association of mTOR with these promoters is regulated by growth signals and sensitive to rapamycin. Together, our observations suggest that mTOR is closely involved in gene regulation at the promoters, which is a conserved mechanism to control RNA polymerase I- and III-dependent genes that are critical for protein synthesis and cell growth.
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PMID:mTOR binds to the promoters of RNA polymerase I- and III-transcribed genes. 2034 49

The mammalian target of rapamycin (mTOR) regulates growth via promoting translation and transcription. Here, employing an mTOR active-site inhibitor WYE-125132 (WYE-132), we have performed quantitative phospho-proteomics and identified a Ser-75-containing phosphopeptide from Maf1, a known repressor of RNA polymerase III (Pol III) transcription. Treatment of cancer cells with WYE-132 or the rapamycin analog CCI-779 led to a rapid loss of the phosphorylation at Ser-75, whereas this effect was not seen in cells treated with cytotoxic agents or unrelated inhibitors. WYE-132-induced Maf1 dephosphorylation correlated with its accumulation in the nucleus and a marked decline in the cellular levels of pre-tRNAs. Depletion of cellular Maf1 via small interfering RNA increased basal pre-tRNA and rendered tRNA synthesis refractory to mTOR inhibitors. Maf1 mutant proteins carrying S75A alone or with S60A, T64A, and S68A (Maf1-S75A, Maf1-4A) progressively enhanced basal repression of tRNA in actively proliferating cells and attenuated amino acid-induced tRNA transcription. Gene alignment revealed conservation of all four Ser/Thr sites in high eukaryotes, further supporting a critical role of these residues in Maf1 function. Interestingly, mTOR inhibition led to an increase in the occupancy of Maf1 on a set of Pol III-dependent genes, with concomitant reduction in the binding of Pol III and Brf1. Unexpectedly, mTORC1 itself was also enriched at the same set of Pol III templates, but this association was not influenced by mTOR inhibitor treatment. Our results highlight a new and unique mode of regulation of Pol III transcription by mTOR and suggest that normalization of Pol III activity may contribute to the therapeutic efficacy of mTOR inhibitors.
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PMID:Requirement of the mTOR kinase for the regulation of Maf1 phosphorylation and control of RNA polymerase III-dependent transcription in cancer cells. 2023 13


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