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)

We have fortuitously identified a nucleotide sequence that decreases expression of a reporter gene in the yeast Saccharomyces cerevisiae 20-fold when inserted into an intron. The primary effect of the insertion is a decrease in pre-mRNA abundance accompanied by the appearance of 3'-truncated transcripts, consistent with premature transcriptional termination and/or pre-mRNA degradation. Point mutations in the cis element relieve the negative effect, demonstrating its sequence specificity. A novel yeast protein, named Nrd1, and a previously identified putative helicase, Sen1, help mediate the negative effect of the cis element. Sen1 is an essential nuclear protein that has been implicated in a variety of nuclear functions. Nrd1 has hallmarks of a heterogeneous nuclear ribonucleoprotein, including an RNA recognition motif, a region rich in RE and RS dipeptides, and a proline- and glutamine-rich domain. An N-terminal domain of Nrd1 may facilitate direct interaction with RNA polymerase II. Disruption of the NRD1 gene is lethal, yet C-terminal truncations that delete the RNA recognition motif and abrogate the negative effect of the cis element nevertheless support cell growth. Thus, expression of a gene containing the cis element could be regulated through modulation of the activity of Nrd1. The recent identification of Nrd1-related proteins in mammalian cells suggests that this potential regulatory pathway is widespread among eukaryotes.
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PMID:Repression of gene expression by an exogenous sequence element acting in concert with a heterogeneous nuclear ribonucleoprotein-like protein, Nrd1, and the putative helicase Sen1. 894 55

A mutation in the rpoA gene (which encodes the alpha subunit of RNA polymerase) that changed the glutamic acid codon at position 261 to a lysine codon decreased the level of expression of a metE-lacZ fusion 10-fold; this decrease was independent of the MetR-mediated activation of metE-lacZ. Glutamine and alanine substitutions at this position are also metE-lacZ down mutations, suggesting that the glutamic acid residue at position 261 is essential for metE expression. In vitro transcription assays with RNA polymerase carrying the lysine residue at codon 261 indicated that the decreased level of metE-lacZ expression was not due to a failure of the mutant polymerase to respond to any other trans-acting factors, and a deletion analysis using a lambda metE-lacZ gene fusion suggested that there is no specific cis-acting sequence upstream of the -35 region of the metE promoter that interacts with the alpha subunit. Our data indicate that the glutamic acid at position 261 in the alpha subunit of RNA polymerase influences the intrinsic ability of the enzyme to transcribe the metE core promoter.
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PMID:The glutamic acid residue at amino acid 261 of the alpha subunit is a determinant of the intrinsic efficiency of RNA polymerase at the metE core promoter in Escherichia coli. 895 1

We have examined the mechanism by which the cAMP-responsive factor CREB stimulates target gene expression following its phosphorylation at Ser-133. Using an in vitro transcription assay, we found that two signals were required for target gene activation: a phospho(Ser-133)-dependent interaction of CREB with RNA polymerase II via the coactivator CBP and a glutamine-rich domain interaction with TFIID via hTAF(II)130. The adenovirus E1A oncoprotein was found to inhibit phospho(Ser-133) CREB activity by binding to CBP and specifically blocking recruitment of RNA Pol II to the promoter. Our results suggest that the recruitment of CBP-RNA Pol II complexes per se is not sufficient for transcriptional activation and that activator-mediated recruitment of TFIID is additionally required for induction of signal-dependent genes.
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PMID:Analysis of a cAMP-responsive activator reveals a two-component mechanism for transcriptional induction via signal-dependent factors. 908 28

Representatives of three distinct classes of mammalian protein domain activating RNA polymerase II were fused to the yeast GAL4p DNA-binding domain. The resulting fusion proteins were tested in the fission yeast Schizosaccharomyces pombe for their ability to activate transcription of different reporter constructs containing GAL4-binding sites in positions close to or far from the TATA box. The acidic-rich activation domain of VP16 stimulates transcription in S.pombe from proximal and distal positions, suggesting that the mechanism of activation is conserved from man to budding and fission yeasts. Unlike in Saccharomyces cerevisiae, the glutamine-rich activation domains of Sp1, Oct1 and Oct2 activate transcription in S. pombe when tested in a proximal TATA box context. Similarly to mammalian cells, these domains are inactive or weakly active when tested in a distal position. Moreover, the proline-rich activation domains of AP-2 and CTF/NF1 display strong transcriptional activities from a TATA box-proximal position, and weak activities when tested in a remote position. Consequently, proline-rich and glutamine-rich activation domains act differently in S.cerevisiae and mammalian cells, but similarly in S.pombe and mammalian cells.
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PMID:Three classes of mammalian transcription activation domain stimulate transcription in Schizosaccharomyces pombe. 931 30

Maximal human immunodeficiency virus type 1 (HIV-1) gene expression requires specific cellular factors in addition to the virus-encoded trans-activator protein Tat and the RNA element TAR. We developed a functional assay, based on transcriptional activation in vitro, to identify these cellular factors. Here, we describe the purification and molecular cloning of CA150, a nuclear protein that is associated with the human RNA polymerase II holoenzyme and is involved in Tat-dependent HIV-1 transcriptional activation. The sequence of CA150 contains an extensive glutamine- and alanine-rich repeat that is found in transcriptional modulators such as GAL11 and SSN6 in Saccharomyces cerevisiae and Zeste in Drosophila melanogaster. Immunodepletion of CA150 abolished Tat trans activation in vitro. Moreover, overexpression of a mutant CA150 protein specifically and dramatically decreased Tat-mediated activation of the HIV-1 promoter in vivo, strongly suggesting a role for CA150 in HIV-1 gene regulation. Immunoprecipitation experiments demonstrated that both CA150 and Tat associate with the RNA polymerase II holoenzyme. Furthermore, we found that functional Tat associates with the holoenzyme whereas activation-deficient Tat mutants do not. Thus, we propose that Tat action is transduced via an RNA polymerase II holoenzyme that contains CA150.
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PMID:CA150, a nuclear protein associated with the RNA polymerase II holoenzyme, is involved in Tat-activated human immunodeficiency virus type 1 transcription. 931 62

The RNA polymerase sigma subunit, sigmaH, of Bacillus subtilis is required for the transcription of genes that are induced in late-growth cultures at high cell density, including genes that function in sporulation. The expression of sigmaH-controlled genes is repressed when nutrient broth sporulation medium (Difco sporulation medium [DSM]) is supplemented with high concentrations of glucose and glutamine (DSM-GG), preferred carbon and nitrogen sources of B. subtilis. Under these conditions, the pH of the DSM-GG medium decreases to approximately 5. Raising the pH by the addition of morpholinepropanesulfonic acid (MOPS) or Tris-HCl (pH 7.5) results in a dramatic increase in the expression of lacZ fusions to sigmaH-dependent promoters. Correspondingly, the level of sigmaH protein was higher in cells of late-growth DSM-GG cultures treated with a pH stabilizer. When sigmaH-dependent gene expression was examined in cells bearing a mutation in abrB, encoding the transition state regulator that negatively controls genes transcribed by the sigmaH form of RNA polymerase, derepression was observed as well as an increase in medium pH. Reducing the pH with acetic acid resulted in repression, suggesting that AbrB was not functioning directly in pH-dependent repression but was required to maintain the low medium pH in DSM-GG. AbrB protein levels were high in late-growth, DSM-GG cultures but significantly lower when the pH was raised by Tris-HCl addition. An active tricarboxylic acid (TCA) cycle was required to obtain maximum derepression of sigmaH-dependent transcription, and transcription of the TCA cycle enzyme gene citB was repressed in DSM-GG but derepressed when the pH was artificially raised. The negative effect of low pH on sigmaH-dependent lacZ expression was also observed in unbuffered minimal medium and appeared to be exerted posttranslationally with respect to spo0H expression. However, the addition of amino acids to the medium caused pH-independent repression of both sigmaH-dependent transcription and spo0H-lacZ expression. These results suggest that spo0H transcription or translation is repressed by a mechanism responding to the availability of amino acids whereas spo0H is posttranslationally regulated in response to external pH.
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PMID:Regulation of Bacillus subtilis sigmaH (spo0H) and AbrB in response to changes in external pH. 935 30

Tat of HIV-2 (Tat-2) requires host cellular factors for optimal function. We show that transactivation by Tat-2 of the HIV promoter requires cis-acting binding sites for Sp1 or Sp1 brought to the promoter via a heterologous system. We demonstrate that an activation domain in Tat-2 consists of one of two potential alpha-helices in the amino-terminal region, the cysteine-rich region, and the core region and that this independent activation domain requires cis-acting Sp1-binding sites for function. Tat-2 interacts with Sp1 in in vitro binding assays, and these interactions require basic residues outside of the Tat-2 activation domain. The regions in Sp1 sufficient for functional synergy with Tat are the Sp1 activation domains, while the DNA-binding region is dispensable. Substitution mutations of a glutamine-rich region in one Sp1 activation domain, which eliminate interactions with a TBP-associated factor, also significantly decrease synergy with Tat. Thus, the functional synergy between Tat-2 and Sp1 localizes to domains in each activator that interact with components of the transcription complex. We suggest that these interactions, rather than direct Tat/Sp1 binding, result in highly processive RNA polymerase II complexes and full-length viral transcripts.
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PMID:Interactions between Tat of HIV-2 and transcription factor Sp1. 940 May 95

Inactivation of transcription factor sigma54, encoded by rpoN (glnF), restores high-temperature growth in Luria-Bertani (LB) medium to strains containing the heat-sensitive cell division mutation ftsZ84. Mutational defects in three other genes involved in general nitrogen control (glnD, glnG, and glnL) also suppress lethal filamentation. Since addition of glutamine to LB medium fully blocks suppression by each mutation, the underlying cause of suppression likely derives from a stringent response to the limitation of glutamine. This model is supported by several observations. The glnL mutation requires RelA-directed synthesis of the nutrient alarmone ppGpp to suppress filamentation. Artificially elevated levels of ppGpp suppress ftsZ84, as do RNA polymerase mutations that reproduce global effects of the ppGpp-induced state. Both the glnF null mutation and an elevated copy number of the relA gene similarly affect transcription from the upstream (pQ) promoters of the ftsQAZ operon, and both of these genetic conditions increase the steady-state level of the FtsZ84 protein. Physiological suppression of ftsZ84 by a high salt concentration was also shown to involve RelA. Additionally, we found that the growth of a glnF or glnD strain on LB medium depends on RelA or supplemental glutamine in the absence of RelA function. These data expand the roles for ppGpp in the regulation of glutamine metabolism and the expression of FtsZ during cell division.
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PMID:Control of ftsZ expression, cell division, and glutamine metabolism in Luria-Bertani medium by the alarmone ppGpp in Escherichia coli. 949 42

The ZFM1 protein is both a transcriptional repressor and identical to the splicing factor SF1. ZFM1 was shown to interact with and repress transcription from the glycine, glutamine, serine, and threonine-rich transcription activation domain of the sea urchin transcription factor, stage-specific activator protein (SSAP). EWS, a human protein involved in cellular transformation in Ewing's sarcoma tumors, contains an NH2-terminal transcriptional activation domain (NTD) which resembles that of SSAP in both amino acid composition and the ability to drive transcription to levels higher than VP16 in most cell types. Here we report that ZFM1 also interacts with EWS in both two-hybrid assays and glutathione S-transferase pull-down experiments. The region on EWS which interacts with ZFM1 maps to 37 amino acids within its NTD. Overexpression of ZFM1 in HepG2 cells represses the transactivation of reporter gene expression driven by Gal4-EWS-NTD fusion protein and this repression correlates with ZFM1 binding to EWS. Furthermore, two proteins, TLS and hTAFII68, which have extensive homology to EWS, also interact with ZFM1. Recently, it was discovered that EWS/TLS/hTAFII68 are each present in distinct TFIID populations and EWS and hTAFII68 were also found to be associated with the RNA polymerase II holoenzyme. The association of ZFM1 with these proteins implies that one normal cellular function for ZFM1 may be to negatively modulate transcription of target genes coordinated by these cofactors.
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PMID:The transcriptional repressor ZFM1 interacts with and modulates the ability of EWS to activate transcription. 966 Jul 65

Spo0A activates transcription in Bacillus subtilis from promoters that are used by two types of RNA polymerase, RNA polymerase containing the primary sigma factor, sigmaA, and RNA polymerase containing a secondary sigma factor, known as sigmaH. The region of sigmaA near positions 356 to 359 is required for Spo0A-dependent promoter activation, possibly because Spo0A interacts with this region of sigmaA at these promoters. To determine if the amino acids in the corresponding region of sigmaH are also important in Spo0A-dependent promoter activation, we examined the effects of single alanine substitutions at 10 positions in sigmaH (201 to 210). Two alanine substitutions in sigmaH, at glutamine 201 (Q201A) and at arginine 205 (R205A), significantly decreased activity from the Spo0A-dependent, sigmaH-dependent promoter spoIIA but did not affect expression from the sigmaH-dependent, Spo0A-independent promoters citGp2 and spoVG. Therefore, promoter activation by Spo0A requires homologous regions in sigmaA and sigmaH. A mutant form of Spo0A, S231F, that suppresses the sporulation defect caused by several amino acid substitutions in sigmaA did not suppress the sporulation defects caused by the Q201A and R205A substitutions in sigmaH. This result and others indicate that different surfaces of Spo0A probably interact with sigmaA and sigmaH RNA polymerases.
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PMID:A region in Bacillus subtilis sigmaH required for Spo0A-dependent promoter activity. 973 8


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