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)

Since the discovery of transcription enhancer sequences in the genomes of the DNA viruses simian virus 40(SV40) and polyoma virus, these elements have been shown to play an important part in the control of both viral and cellular gene expression. Enhancer elements act in cis to increase the amount of RNA produced from linked genes in a manner largely independent of distance and orientation. The mechanisms by which enhancers act are not understood; in particular, it is not known whether the enhancer-dependent increase in the level of stable RNA reflects an increase in the rate of transcription. To address this question, we have used an in vitro nuclear transcription assay to examine the effect of the SV40 enhancer on transcription of cloned human beta-globin genes transiently introduced into HeLa cells. We show here that the SV40 enhancer acts at least in part to increase the number of RNA polymerase II molecules transcribing the linked gene.
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PMID:Simian virus 40 enhancer increases number of RNA polymerase II molecules on linked DNA. 298 13

We examined the structural and functional properties of a human H3 histone gene promoter. The complete nucleotide sequence of an H3 structural gene and 515 nucleotides of 5' and 100 nucleotides of 3' flanking sequences were determined. The upstream region of this cell cycle dependent H3 histone gene, designated pST519, contains consensus sequences typical of genes transcribed by RNA polymerase II. To address promoter function directly, we determined the capability of the 5' flanking sequences to direct the transcription of two genes which are not functionally or structurally related. Fusion genes were constructed using the 5' flanking sequences of this human H3 histone gene and either human beta-globin or bacterial chloramphenicol acetyltransferase (CAT) coding sequences. Both of these fusion genes were expressed when transfected into HeLa cells. Under control of the pST519 histone gene promoter, a beta-globin mRNA transcript was initiated at the appropriate H3 (bp) enhancer, inserted upstream from the histone promoter in both fusion constructs, increased levels of beta-globin and CAT expression. Expression of the pST519 H3 histone gene in COS cells in the absence of the SV40 72-bp enhancer confirmed that the sequences required for promoting transcription reside within the 750-bp 5' flanking sequences and that the exogenous enhancer facilitates, but is not a prerequisite for, transcription. Enhancer-facilitated expression of a cell cycle dependent human H4 histone gene was also observed following transfection into mouse L cells and indicates that the regulatory sequences of human histone genes and transcription factors of mouse cells are compatible.
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PMID:Enhancer-facilitated expression of prokaryotic and eukaryotic genes using human histone gene 5' regulatory sequences. 301 46

Enhancer-dependent transcription in enteric bacteria depends upon an activator protein that binds DNA far upstream from the promoter and an alternative sigma factor (sigma 54) that binds with the core RNA polymerase at the promoter. In the photosynthetic bacterium Rhodobacter capsulatus, the NtrB and NtrC proteins (RcNtrB and RcNtrC) are putative members of a two-component system that is novel because the enhancer-binding RcNtrC protein activates transcription of sigma 54-independent promoters. To reconstitute this putative two-component system in vitro, the ReNtrB protein was overexpressed in Escherichia coli and purified as a maltose-binding protein fusion (MBP-RcNtrB). MBP-RcNtrB autophosphorylates in vitro to the same steady state level and with the same stability as the Salmonella typhimurium NtrB (StNtrB) protein but at a lower initial rate. MBP-RcNtrB autophosphorylates the S.typhimurium NtrC (St-NtrC) and RcNtrC proteins in vitro. The enteric NtrC protein is also phosphorylated in vivo by RcNtrB because plasmids that encode either RcNtrB or MBP-Rc-NtrB activate transcription of an NtrC-dependent nifL-lacZ fusion. The rate of phosphotransfer to RcNtrC and autophosphatase activity of phosphorylated RcNtrC (RcNtrC---P) are comparable to the StNtrC protein. However, the RcNtrC protein appears to be a specific RcNtrB P phosphatase since RcNtrC is not phosphorylated by small molecular weight phosphate compounds or by the StNtrB protein. RcNtrC forms a dimer in solution, and RcNtrC - P binds the upstream tandem binding sites of the g1nB promoter 4-fold better than the unphos-phorylated RcNtrC protein, presumably due to oligomerization of RcNtrC -P. Therefore, the R. capsulatus NtrB and NtrC proteins form a two-component system similar to other NtrC-like systems, where specific Rc- NtrB phosphotransfer to the RcNtrC protein results in increased oligoinerization at the enhancer but with subsequent activation of a sigma 54-independent promoter.
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PMID:In vitro reconstitution and characterization of the Rhodobacter capsulatus NtrB and NtrC two-component system. 862 57

Gene transcription is governed by a set of basal transcription machineries and gene-specific factors. Eukaryotic RNA polymerases alone can not direct specific transcription, but need associated factors, namely general transcription factors (GTFs). The basal transcription machineries composed of RNA polymerase and GTFs bind to a promoter and govern efficient and correct transcription for constitutive gene expression. Protein-coding genes are transcribed by RNA polymerase (Pol) II whereas Pol I and Pol III synthesize ribosomal RNA and various small RNAs, respectively. Enhancer is another class of cis-element for Pol II to which transcription regulatory factors bind. Those factors are involved in inducible, repressive, and tissue-specific gene expressions via binding to their target sequences. Regulatory factors have multiple structural motifs and interact with basal machineries directly or indirectly (using mediators) in addition to DNA. Many transcription factors are known to regulate nervous system-specific gene expression, which include bHTH, bHLH, basic leucine zipper, and zinc finger factors and prorine-rich activators. These factors, some of which belong to a neural silencer factor, play roles in neural development, establishment of memory and learning, and expression of nervous system-specific proteins.
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PMID:Mechanisms of transcriptional regulation and neural gene expression. 911 25

Enhancer-dependent transcription in bacteria requires the alternative transcription factor sigma N (sigma 54), which forms an RNA polymerase holoenzyme that binds promoters as a transcriptionally inactive complex. We have examined the structure of sigma N by circular dichroism (CD) analysis. The sigma N protein and its domains are well structured in the absence of the core RNA polymerase subunits or promoter DNA. Denaturation of sigma N by temperature as followed by changes in CD shows a concomitant loss of secondary and tertiary structures with a melting temperature of 36 degrees C. The secondary structure displays a two-state melting curve with a second Tm of 85 degrees C. The amino-terminal Region I activation domain together with the acidic Region II does not contribute to the two-state melting. In marked contrast, the integrity of the C-terminal DNA-binding domain is required for the two-state melting. Measurements of pKb also demonstrated that a C-terminal part of sigma N, but not regions I or I + II, is required for the structural integrity of sigma N at high pH. Measurements of pKa suggested that alpha-helical structures are important in sigma N for the establishment of tertiary structural elements. The tertiary structure near ultraviolet CD signals of sigma N do not require regions I or I + II but were strongly diminished by C-terminal truncation of sigma N. Promoter DNA binding resulted in a conformational change in sigma N, permitting the determination of a binding constant. A typical B-DNA conformation was adopted by the promoter DNA. Implications for the modular domain organization of sigma N, the function of C-terminal sequences, and domain communication and its role in activation of transcription are discussed.
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PMID:Analysis of the architecture of the transcription factor sigma N (sigma 54) and its domains by circular dichroism. 917 57

1. Dissociated rat superior cervical ganglion (SCG) neurons have been shown to possess a hyperpolarization-activated inwardly rectifying chloride current. The current was not altered by changes in external potassium concentration, replacing external cations with NMDG (N-methyl-D-glucamine) or by addition of 10 mM caesium or barium ions. 2. The reversal potential of the current was altered by changing external anions. The anion selectivity of the current was Cl- > Br- > I- > cyclamate. All substituted permeant anions also blocked the current. 3. The current was blocked by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid), 9AC (anthracene-9-carboxylic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) but was unaffected by SITS (4-acetamido-4'-isothiocyanatostilbene- 2,2'-disulphonic acid) and niflumic acid. The effective blockers were voltage dependent; DIDS and NPPB were more effective at depolarized potentials while 9AC was more effective at hyperpolarized potentials. 4. The current was enhanced by extracellular acidification and reduced by extracellular alkalinization. Reducing external osmolarity was without effect in conventional whole-cell recording but enhanced current amplitude in those perforated-patch recordings where little current was evident in control external solution. 5. The current in SCG neurons was blocked by external cadmium and zinc. ClC-2 chloride currents expressed in Xenopus oocytes were also sensitive to block by these divalent ions and by DIDS but the sensitivity of ClC-2 to block by cadmium ions was lower than that of the current in SCG neurons. 6. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed the presence of mRNA for ClC-2 in SCG neurons but not in rat cerebellar granule cells which do not possess a hyperpolarization-activated Cl- current. 7. The data suggest that ClC-2 may be functionally expressed in rat SCG neurons. This current may play a role in regulating the internal chloride concentration in these neurons and hence their response to activation of GABAA receptors.
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PMID:Characterization of the hyperpolarization-activated chloride current in dissociated rat sympathetic neurons. 950 29

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein of 469 residues that activates transcription by sigma(54)-holoenzyme. A region of its transcriptional activation (central) domain that is highly conserved among homologous activators of sigma(54)-holoenzyme-residues 206-220-is essential for interaction with this RNA polymerase: it is required for contact with the polymerase and/or for coupling the energy from ATP hydrolysis to a change in the conformation of the polymerase that allows it to form transcriptionally productive open complexes. Several mutant NtrC proteins with amino acid substitutions in this region, including NtrC(A216V) and NtrC(G219K), have normal ATPase activity but fail in transcriptional activation. We now report that other mutant forms carrying amino acid substitutions at these same positions, NtrC(A216C) and NtrC(G219C), are capable of activating transcription when they are not bound to a DNA template (non-DNA-binding derivatives with an altered helix-turn-helix DNA-binding motif at the C terminus of the protein) but are unable to do so when they are bound to a DNA template, whether or not it carries a specific enhancer. Enhancer DNA remains a positive allosteric effector of ATP hydrolysis, as it is for wild-type NtrC but, surprisingly, appears to have become a negative allosteric effector for some aspect of interaction with sigma(54)-holoenzyme. The conserved region in which these amino acid substitutions occur (206-220) is equivalent to the Switch I region of a large group of purine nucleotide-binding proteins. Interesting analogies can be drawn between the Switch I region of NtrC and that of p21(ras).
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PMID:"Switch I" mutant forms of the bacterial enhancer-binding protein NtrC that perturb the response to DNA. 1055 87

We have isolated from the rat cerebellum cDNA library a complementary DNA encoding a new member of the tandem pore K(+) channel family. Its amino acid sequence shares 54% identity with that of TASK-1, but less than 30% with those of TASK-2 and other tandem pore K(+) channels (TWIK, TREK, TRAAK). Therefore, the new clone was named TASK-3. Reverse transcriptase-polymerase chain reaction analysis showed that TASK-3 mRNA is expressed in many rat tissues including brain, kidney, liver, lung, colon, stomach, spleen, testis, and skeletal muscle, and at very low levels in the heart and small intestine. When expressed in COS-7 cells, TASK-3 exhibited a time-independent, noninactivating K(+)-selective current. Single-channel conductance was 27 pS at -60 mV and 17 pS at 60 mV in symmetrical 140 mM KCl. TASK-3 current was highly sensitive to changes in extracellular pH (pH(o)), a hallmark of the TASK family of K(+) channels. Thus, a change in pH(o) from 7.2 to 6.4 and 6.0 decreased TASK-3 current by 74 and 96%, respectively. Mutation of histidine at position 98 to aspartate abolished pH(o) sensitivity. TASK-3 was blocked by barium (57%, 3 mM), quinidine (37%, 100 microM), and lidocaine (62%, 1 mM). Thus, TASK-3 is a new member of the acid-sensing K(+) channel subfamily (TASK).
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PMID:TASK-3, a new member of the tandem pore K(+) channel family. 1073 76

Enhancer-dependent activator proteins, which act upon the bacterial RNA polymerase containing the sigma54 promoter specificity factor, belong to the AAA superfamily of ATPases. Activator-sigma54 contact is required for the sigma54-RNAP to isomerize and engage the DNA template for transcription. How ATP hydrolysis is used to trigger changes in sigma54-RNA polymerase and promoter DNA that lead to DNA opening is poorly understood. Here, band shift and footprinting assays were used to investigate the DNA binding activities of sigma54 and sigma54-RNA polymerase in the presence of the activator protein PspF bound to poorly hydrolysable analogues of ATP and the ATP hydrolysis transition-state analogue ADP.AlFx. Results show that different nucleotide-bound forms of PspF can change the interactions between sigma54, sigma54-RNA polymerase, and a DNA fork junction structure present within closed promoter complexes. This provides evidence that in the activation transduction pathway, several functional states of the activator, prior to ATP hydrolysis, can serve to alter the fork junction binding activity of sigma54 and sigma54-RNA polymerase that precede full DNA opening. A sequential set of nucleotide-dependent transitions in sigma54-RNA polymerase promoter complexes needed for productive open complex formation may therefore depend upon different nucleotide-bound forms of the activator.
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PMID:Nucleotide-dependent triggering of RNA polymerase-DNA interactions by an AAA regulator of transcription. 1264 85

Down-regulation of T-type Ca channel current and mRNA occurs following differentiation of Y79 retinoblastoma cells. To understand how the decrease in expression is linked to cell differentiation, we examined transcriptional regulation of the Cav3.1 Ca channel gene, CACNA1G. We identified two putative promoters (A and B) in 1.3 kb of cloned genomic DNA. Reverse transcriptase-polymerase chain reaction and 5' rapid amplification of cDNA ends-polymerase chain reaction analyses demonstrated that two transcripts with different 5' untranslated regions are generated by different transcription start sites, with promoter A favoured in undifferentiated cells and promoter B favoured in differentiated cells. Functional analyses of the promoter sequence revealed that both promoters are active. Enhancer and repressor sequences were identified upstream of promoter A and B, respectively. These results suggest that the down-regulation of alpha1G mRNA in differentiated Y79 cells is mediated primarily by decreased activity of promoter A, which could occur in conjunction with repression of the activity of promoter B. The decrease in T-type Ca channel expression in Y79 cells may be an essential signal affecting phenotypic maturation and expression of other ion channel subtypes in the differentiated cells.
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PMID:Regulation of alpha1G T-type calcium channel gene (CACNA1G) expression during neuronal differentiation. 1275 79


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