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)

Efficient transcription elongation by RNA polymerase I (Pol I) requires a specific Pol I-associated factor, termed TIF-IC. Here we show that TFIIS, a factor that has previously been shown to promote read-through past many types of blocks to elongation by RNA polymerase II, also enhances Pol I-directed transcription elongation. In a reconstituted transcription system containing purified proteins, TFIIS stimulates Pol I transcription by increasing the overall rate of RNA chain elongation. As with Pol II, ternary Pol I complexes cleave the 3' end of the nascent transcripts in response to TFIIS. The truncated RNAs remain bound to the template, are subject to pyrophosphorolysis, and can be chased into longer transcripts. Moreover, we show by immunoprecipitation and specific affinity chromatography that TFIIS physically interacts with Pol I. The results suggest that nascent transcript cleavage by TFIIS or a TFIIS-related factor may be a general mechanism by which both Pol I and Pol II can bypass transcriptional impediments.
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PMID:TFIIS binds to mouse RNA polymerase I and stimulates transcript elongation and hydrolytic cleavage of nascent rRNA. 887 42

The protein Tat is encoded by the HIV-1 genome and is essential for viral replication because of its activation of viral transcription. Tat enhances the ability of RNA polymerase II (Pol II) to move long distances down the DNA through a poorly understood mechanism that involves its binding the to the 5' end of the nascent HIV-1 transcript. It has been suggested that the stimulation of transcript elongation by conventional DNA-binding activators may involve phosphorylation of the carboxy-terminal domain (CTD) of Pol II by the transcription factor TFIIH through the associated CAK kinase. Here we show that Tat-enhanced HIV-1 transcription in vitro requires both TFIIH and the CTD of Pol II. In addition, Tat, through its activation domain, both interacts with a functional TFIIH-containing complex and stimulates phosphorylation of a CTD-containing substrate by the TFIIH kinase. Under conditions that jointly restrict transcriptional elongation and TFIIH-mediated CTD phosphorylation, Tat stimulates both these activities. Furthermore, RNA synthesis is required for Tat to stimulate phosphorylation of the CTD when it is part of an initiation complex, as expected from Tat's interaction with viral transcripts. Thus, stimulation of Pol II elongation by Tat may involve direct effects on TFIIH-mediated CTD phosphorylation.
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PMID:Enhanced processivity of RNA polymerase II triggered by Tat-induced phosphorylation of its carboxy-terminal domain. 893 26

Nonhistone proteins 6A and 6B (NHP6A/B) are nonsequence-specific DNA-binding proteins from Saccharomyces cerevisiae that are related structurally and functionally to the mammalian high mobility group proteins 1 and 2. These DNA architectural proteins distort DNA structure severely and have been shown to promote assembly of specialized recombination complexes. Here we show that the yeast NHP6A/B proteins are required for the induction of a subset of genes transcribed by RNA polymerase II (pol II). Activation of the CUP1, CYC1, GAL1, and DDR2 genes was decreased or abolished completely in the delta nhp6A/B strain. No significant change in basal expression was observed for any of the 10 genes examined. Analysis of chimeric gene constructs localized the regions dependent on NHP6A/B to be primarily at the core promoters, although the GAL1 UAS also requires NHP6A/B for activity. In vitro, NHP6A stimulated transcription by pol II at the GAL1 promoter three- to fivefold above the level of activation by GAL4-VP16 alone. Gel mobility shift assays showed that NHP6A promotes the formation of a complex with TBP and TFIIA at the TATA box that has enhanced affinity for TFIIB.
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PMID:Yeast HMG proteins NHP6A/B potentiate promoter-specific transcriptional activation in vivo and assembly of preinitiation complexes in vitro. 894 17

The herpes simplex virus thymidine kinase gene (HSV-TK) in combination with ganciclovir (GCV), is currently being used in gene therapy-based clinical trials for cancer treatment. Its therapeutic effect is based on a "bystander effect" whereby HSV-TK gene-modified tumor cells are toxic to nearby unmodified tumor cells when exposed to the antiviral drug GCV. We have recently hypothesized that the in vivo mechanism of this bystander effect is due to alterations in the tumor microenvironment in response to release of cytokines and an infiltration of leukocytes after treatment with HSV-TK gene-modified tumor cells and GCV, which results in tumor regression. Expression of B7, a recently identified costimulatory molecule that is important for T-cell stimulation, has been shown to be modulated by stimulatory cytokines interferon-gamma, tumor necrosis factor-alpha, and inhibited by interleukin-10. In the present study, we investigated whether the cytokines released after HSV-TK and GCV treatment could include the expression of the costimulatory molecules B7-1 and B7-2 and the adhesion molecule (ICAM)-1 in the tumor. Furthermore, we investigated whether this altered environment affected the antitumor properties of host lymphocytes. An in vitro model was developed to establish the effects of HSV-TK gene-modified tumor cells and GCV on tumor infiltrating cells. The murine macrophage cell line (IC21) was exposed to either supernatants or cell lysates collected from a mixture of HSV-TK-transduced (KBALB-STK) and non-transduced (KBALB) murine fibrosarcoma tumor cells previously exposed to GCV (experimental). Immunohistochemical analysis showed a significant expression (P < .0001) of B7-1 and B7-2 post exposure of IC21 cells to either supernatant or lysate. In contrast, the level of expression in IC21 cells exposed to the control lysate or supernatant remained unchanged for B7-1 and B7-2. In vivo analysis for B7-1 and B7-2 expression by immunohistochemistry in tumor tissues from experimental mice receiving HSV-TK gene-modified tumor cells and GCV treatment showed a significant expression of B7.1 (35%, P < .0001) and B7.2 (38.2%, P < .0001) on tumor-infiltrating mononuclear cells. In contrast, tumor-bearing control animals showed low levels of B7-2 expression (5.8%), whereas B7-1 was undetectable, as confirmed by reverse-transcriptase polymerase chain reaction. In addition, a significant up-regulation of ICAM expression (50%) on tumor tissues was observed in the experimental group (P = .0317) as compared with the control group (25%). Furthermore, T cells isolated from experimental mice showed a significant in vitro proliferative response (p = .0202) when exposed to syngeneic tumor cells as compared with the control group. These data demonstrated that the use of HSV-TK gene-modified tumor cells and GCV as a suicide gene in the treatment of an intraperitoneal tumor resulted in the expression of the B7 costimulatory molecules and ICAM-1 adhesion molecule and enhanced proliferative response of host T cells. These findings help to understand the mechanism of tumor cell killing in vivo using HSV-TK gene-modified tumor cells.
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PMID:Expression of costimulatory molecules: B7 and ICAM up-regulation after treatment with a suicide gene. 898 40

A new ribosomal RNA promoter element with a functional role similar to the RNA polymerase II initiator (Inr) was identified. This sequence, which we dub the ribosomal Inr (rInr) is unusually conserved, even in normally divergent RNA polymerase I promoters. It functions in the recruitment of the fundamental, TATA-binding protein (TBP)-containing transcription factor, TIF-IB. All upstream elements of the exceptionally strong Acanthamoeba castellanii ribosomal RNA core promoter, to within 6 base pairs of the transcription initiation site (tis), can be deleted without loss of specific transcription initiation. Thus, the A. castellanii promoter can function in a manner similar to RNA polymerase II TATA-less promoters. Sequence-specific photo-cross-linking localizes a 96-kDa subunit of TIF-IB and the second largest RNA polymerase I subunit (A133) to the rInr sequence. A185 also photo-cross-links when polymerase is stalled at +7.
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PMID:Identification of previously unrecognized common elements in eukaryotic promoters. A ribosomal RNA gene initiator element for RNA polymerase I. 901 45

Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs). Unlike class II- and III-specific TBP-TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB/SL1 exhibits a pronounced selectivity for its homologous promoter. As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAFIs) and compared the amino acid sequences of the murine TAFIs with their human counterparts. The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAFIs, demonstrating that differences in the primary structure of human and mouse TAFIs do not dramatically alter the network of protein-protein contacts responsible for assembly of the multimeric complex. Thus, primate vs. rodent promoter selectivity mediated by the TBP-TAFI complex is likely to be the result of cumulative subtle differences between individual subunits that lead to species-specific properties of RNA polymerase I transcription.
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PMID:Cloning of murine RNA polymerase I-specific TAF factors: conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1. 905 Aug 47

Receptor tyrosine kinases (RTK) play an important role in the signal transduction of normal and malignant cells. There are different families of RTKs which are mainly characterized by differences in the ligang-binding extracellular domains. Axl (or UFO/Ark) is the first member of a new class of RTK with two fibronectin type III domains and two immunoglobulin-like domains present at the extracellular domain. The axl-gene has been isolated by means of gene transfection studies using DNA of patients with chronic myelogeneous leukemia. For a previous and the present study, we used a sensitive reverse-transcriptase polymerase chain reaction assay to detect axl's mRNA in cells from normal and malignant hematopoietic tissue. Axl's mRNA expression was mainly detected in myelo-monocytic cells, whereas much weaker transcription was seen in lymphatic cells and in lymphatic leukemias. In normal bone marrow, axl was heavily transcribed in marrow stromal cells. Further, we analysed Axl protein expression using monoclonal antibody M50 in peripheral stem cell harvests; in most harvests, no co-expression of CD34 and Axl was detected. However, in one patient with AML in complete remission, Axl was co-expressed on 80% of the CD34-positive population. These data show that axl is preferentially expressed in monocytes and stromal cells. Furthermore, a fraction of CD34-positive progenitor cells may express Axl. The exact mechanism for transformation of myeloid progenitor cells through Axl, however, remains to be determined.
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PMID:Recent progress on the role of Axl, a receptor tyrosine kinase, in malignant transformation of myeloid leukemias. 913 Jun 17

The cyclin-dependent kinase (CDK)-activating kinase CAK has been proposed to function in the control of cell cycle progression, DNA repair and RNA polymerase II (pol II) transcription. Most CAK exists as complexes of three subunits: CDK7, cyclin H (CycH) and MAT1. This tripartite CAK occurs in a free form and in association with 'core' TFIIH, which functions in both pol II transcription and DNA repair. We investigated the substrate specificities of different forms of CAK. Addition of the MAT1 subunit to recombinant bipartite CDK7-CycH switched its substrate preference to favour the pol II large subunit C-terminal domain (CTD) over CDK2. We suggest that the MAT1 protein, previously shown to function as an assembly factor for CDK7-CycH, also acts to modulate CAK substrate specificity. The substrate specificities of natural TFIIH and free CAK were also compared. TFIIH had a strong preference for the CTD over CDK2 relative to free CAK. TFIIH, but not free CAK, could efficiently hyperphosphorylate the CTD. In the context of TFIIH, the kinase also acquired specificity for the general transcription factors TFIIE and TFIIF which were not recognized by free CAK. We conclude that the substrate preference of the CDK7-CycH kinase is governed by association with both MAT1 and 'core' TFIIH.
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PMID:Regulation of CDK7 substrate specificity by MAT1 and TFIIH. 913 Jul 9

The retinoblastoma susceptibility gene product pRb restricts cellular proliferation by affecting gene expression by all three classes of nuclear RNA polymerases. To elucidate the molecular mechanisms underlying pRb-mediated repression of ribosomal DNA (rDNA) transcription by RNA polymerase I, we have analyzed the effect of pRb in a reconstituted transcription system. We demonstrate that pRb, but not the related protein p107, acts as a transcriptional repressor by interfering with the assembly of transcription initiation complexes. The HMG box-containing transcription factor UBF is the main target for pRb-induced transcriptional repression. UBF and pRb form in vitro complexes involving the C-terminal part of pRb and HMG boxes 1 and 2 of UBF. We show that the interactions between UBF and TIF-IB and between UBF and RNA polymerase I, respectively, are not perturbed by pRb. However, the DNA binding activity of UBF to both synthetic cruciform DNA and the rDNA promoter is severely impaired in the presence of pRb. These studies reveal another mechanism by which pRb suppresses cell proliferation, namely, by direct inhibition of cellular rRNA synthesis.
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PMID:Mechanism of repression of RNA polymerase I transcription by the retinoblastoma protein. 923 80

Genes for the Tfb2, Tfb3, and Tfb4 subunits of yeast RNA polymerase transcription factor IIH (TFIIH) are described. All three genes are essential for cell viability, and antibodies against Tfb3 specifically inhibit transcription in vitro. A C-terminal deletion of Tfb2 caused a defect in nucleotide excision repair, as shown by UV sensitivity of the mutant strain and loss of nucleotide excision repair activity in cell extracts (restored by the addition of purified TFIIH). An interaction between Tfb3 and the Kin28 subunit of TFIIH was detected by the two-hybrid approach, consistent with a role for Tfb3 in linking kinase and core domains of the factor. The deduced amino acid sequence of Tfb2 is similar to that of the 52-kDa subunit of human TFIIH, while Tfb3 is identified as a RING finger protein homologous to the 36-kDa subunit of murine CAK (cyclin-dependent kinase activating kinase) and to the 32-kDa subunit of human TFIIH. Tfb4 is homologous to p34 of human TFIIH and is identified as the weakly associated 37-kDa subunit of the yeast factor. These and other findings reveal a one-to-one correspondence and high degree of sequence similarity between the entire set of yeast and human TFIIH polypeptides.
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PMID:Genes for Tfb2, Tfb3, and Tfb4 subunits of yeast transcription/repair factor IIH. Homology to human cyclin-dependent kinase activating kinase and IIH subunits. 923 28


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