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 ada gene of Escherichia coli K-12 encodes the 39-kDa Ada protein, which consists of two domains joined by a hinge region that is sensitive to proteolytic cleavage in vitro. The amino-terminal domain has a DNA methyltransferase activity that repairs the S-diastereoisomer of methylphosphotriesters while the carboxyl-terminal domain has a DNA methyltransferase activity that repairs O6-methylguanine and O4-methylthymine lesions. Transfer of a methyl group to Cys-69 by repair of a methylphosphotriester lesion converts Ada into a transcriptional activator of the ada and alkA genes. Activation of ada, but not alkA, requires elements contained within the carboxyl-terminal domain of Ada. In addition, physiologically relevant concentrations of the unmethylated form of Ada specifically inhibit methylated Ada-promoted ada transcription both in vitro and in vivo and it has been suggested that this phenomenon plays a pivotal role in the down-regulation of the adaptive response. A set of site-directed mutations were generated within the hinge region, changing the lysine residue at position 178 to leucine, valine, glycine, tyrosine, arginine, cysteine, proline, and serine. All eight mutant proteins have deficiencies in their ability to activate ada transcription in the presence or absence of a methylating agent but are proficient in alkA activation. AdaK178P (lysine 178 changed to proline) is completely defective for the transcriptional activation function of ada while it is completely proficient for transcriptional activation of alkA. In addition, AdaK178P possesses both classes of DNA repair activities both in vitro and in vivo. Transcriptional activation of ada does not occur if both the amino- and carboxyl-terminal domains are produced separately within the same cell. The mutation at position 178 might interfere with activation of ada transcription by changing a critical contact with RNA polymerase, by causing a conformational change of Ada, or by interfering with the communication of conformational information between the amino- and the carboxyl-terminal domains. These results indicate that the hinge region of Ada is important for ada but not alkA transcription and further support the notion that the mechanism(s) by which Ada activates ada transcription differs from that by which it activates transcription at alkA.
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PMID:Alteration of lysine 178 in the hinge region of the Escherichia coli ada protein interferes with activation of ada, but not alkA, transcription. 786 1

Transcription of the 72kb linear double-stranded DNA genome of coliphage N4 is carried out by the sequential activity of three different RNA polymerases. Early and middle viral transcripts are synthesized by two phage-coded RNA polymerases while late transcription is carried out by the Escherichia coli sigma 70-RNA polymerase. We have determined the sequences and sites of initiation of several N4 late transcripts; N4 late promoters share weak homology with the E. coli sigma 70 promoter consensus sequence. Indeed, N4 late promoters are weak templates for the host enzyme. We present evidence that the phage-coded, single-stranded DNA-binding protein (N4SSB), a protein that is required for phage DNA replication and recombination and does not bind with sequence specificity to DNA, is the activator of E. coli RNA polymerase at late N4 promoters. Models for the mechanism of action of N4SSB as a transcriptional activator are discussed.
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PMID:The bacteriophage N4-coded single-stranded DNA-binding protein (N4SSB) is the transcriptional activator of Escherichia coli RNA polymerase at N4 late promoters. 787 67

We report expression of the wt1 (Wilms' tumor) gene by cultured human melanoma cells. Using RNA polymerase chain reaction analysis, wt1 transcripts were detected in 7 of 9 melanoma cell lines but not in 5 normal melanocyte strains. In Northern blot analysis, steady-state wt1 mRNA levels were found in 2 of 4 melanoma lines but not in normal melanocytes. Sequence analysis of the wt1 cDNA expressed by melanoma cell line WM 902-B revealed the presence of 4 previously published splice variants but no evidence for mutations in the coding region. Previous work has shown that WT1 modulates transcription after binding to the early growth response (EGR)-1 sites present in the platelet-derived growth factor (PDGF)-A chain promoter; the PDGF-A chain gene is known to be expressed by various melanoma cell lines. Based on these findings, we studied the relationship of wt1 and PDGF-A chain gene expression in melanoma cell lines. Co-expression of the wt1 and the PDGF-A chain genes was observed in 2 melanoma cell lines with mutated p53 but not in 2 melanoma cell lines with wild-type p53; this result is consistent with a previous report showing that, in the context of absent or mutated p53, WT1 acts as a transcriptional activator, whereas in the presence of wild-type p53 it acts as a repressor.
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PMID:Expression of the wt1 Wilms' tumor gene by normal and malignant human melanocytes. 792 8

The differential localization of proteins in the Caulobacter predivisional cell leads to the formation of two distinct progeny cells: a motile swarmer cell and a sessile stalked cell. Pole-specific transcription in the predivisional cell is one mechanism responsible for protein localization. Here we show that the sigma 54 transcriptional activator FlbD, which activates swarmer pole-specific transcription of a subset of late flagellar genes, is also capable of functioning as a pole-specific repressor of the early flagellar fliF operon. DNase I footprinting and methylation interference assays indicate that FlbD binds to regions of the fliF promoter at regions that would be likely to interfere with the binding of RNA polymerase. A mutation that abolishes FlbD binding results in up to a fourfold increase in fliF promoter expression. This mutation alters both the spatial and temporal pattern of fliF expression resulting in the inappropriate expression of the fliF operon in the swarmer pole of the predivisional cell. These results demonstrate that FlbD represses early flagellar gene expression in the swarmer pole of the Caulobacter predivisional cell. This is the first instance in which a protein specifically involved in pole-specific repression has been identified in Caulobacter. The restriction of FlbD activity to the swarmer pole accomplishes two regulatory missions by simultaneously activating late flagellar gene expression and repressing early flagellar genes.
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PMID:A sigma 54 transcriptional activator also functions as a pole-specific repressor in Caulobacter. 795 61

The expression of nif genes in Rhodobacter capsulatus depends on the two regulatory genes, rpoN and nifA, encoding a nif-specific alternative sigma factor of RNA polymerase and a nif-specific transcriptional activator, respectively. The expression of the rpoN gene itself is also RPON/NIFA dependent. In order to better characterize the regulation of nif gene induction, chromosomal nifH-, rpoN-, nifA1- and nifA2- lacZ fusions were constructed and the expression of these different nif-lacZ fusions was determined under photoheterotrophic conditions at different starting ammonium concentrations. The two nifA genes were found to be induced first, followed by nifH and finally by rpoN upon weak, medium and strong nitrogen starvation, respectively. This induction profile and the correlation between the expression of the different nif genes suggested that nifA1 expression is the limiting factor for nif gene induction. This hypothesis was tested by construction of different nifA1 overexpressing mutants. Contrary to the current model of nif gene expression in R. capsulatus, which predicted constitutive nif gene expression in such mutants, a strong repression of nifH and rpoN was found at high ammonium concentration. The low nifH expression under these conditions is unaffected by nifA2 and is not increased in a ntrC mutant, ruling out any role of NTRC as a mediator of this repression. This finding implies an additional, so far unidentified, regulation by fixed nitrogen in R. capsulatus. Changing the expression level of rpoN indicated that low levels of RPON are already sufficient for full nifH induction. The nifA1 and rpoN expression mutants were also tested for diazotrophic growth. Similar generation times were determined for the mutants and for the wild type, but diazotrophic growth of the nifA1 over-expressing ntrC mutant RCM14 did not start until after a prolonged lag phase of two to three days.
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PMID:nif gene expression studies in Rhodobacter capsulatus: ntrC-independent repression by high ammonium concentrations. 796 8

The prokaryotic enhancer-binding protein NIFA is a multidomain transcriptional activator that catalyzes the formation of open complexes at nitrogen fixation (nif) promoters by a specialized form of RNA polymerase containing sigma 54. The NIFA protein from Klebsiella pneumoniae consists of three domains: the N-terminal domain of unknown function; the central catalytic domain, which is sufficient for transcriptional activation; and the C-terminal DNA-binding domain. Purified fusion proteins between maltose-binding protein (MBP) and NIFA deleted of its N-terminal domain (MBP-delta N-NIFA) or its C-terminal domain (MBP-NIFA-delta C) activated transcription from the K. pneumoniae nifH promoter both in vitro and in vivo. We previously showed that the same was true for a fusion between MBP and the central domain of NIFA. These results indicate that NIFA is sufficiently modular for all fusions carrying its catalytic domain to be active. Unexpectedly, however, simple predictions regarding the location of determinants of the heat lability and insolubility of NIFA, which were based on previous studies of its isolated central and C-terminal domains, were not borne out. Contrary to a previous report from this laboratory, we found that the in vitro start site of transcription for the K. pneumoniae nifH operon could be either of two adjacent G residues, as others had reported in vivo. This was true independent of the activator, i.e., with MBP-NIFA and MBP-delta N-NIFA and with the homologous activator NTRC. When open complexes were formed with GTP as the activating nucleotide, the upstream G residue was probably as a consequence of initiation of transcription.
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PMID:In vitro studies of the domains of the nitrogen fixation regulatory protein NIFA. 800 17

PobR is a transcriptional activator required for the expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase. The pobA and pobR genes are divergently transcribed and separated by 134 bp in the Acinetobacter calcoaceticus chromosome. Primer extension analysis revealed that the pobA transcript begins 22 bp upstream from the structural gene and the pobR transcript begins 69 bp upstream from the regulatory gene. This arrangement requires superimposition of the -10 base pair and -35 base pair RNA polymerase-binding sites for the respective genes. Expression of a pobR-lacZ fusion was found to be repressed three- to fourfold by pobR when the functional gene was carried in trans on a plasmid. The pobR gene was placed under control of a lac promoter in an expression vector, and the recombinant plasmid inducibly expressed high levels of PobR in Escherichia coli. Cell extracts containing this protein were used to conduct gel mobility shift analyses. PobR binds specifically to DNA in the pobA-pobR intergenic region, and this binding does not appear to be influenced by p-hydroxybenzoate, the inducer of pobA expression. DNase I footprinting indicates that the DNA-binding site for PobR extends from about 10 bp to about 45 bp downstream from the site of the beginning of the pobR transcript. Within this putative operator is a region of inverted symmetry. Evidently, interaction of the inducer with the PobR-operator complex triggers elevated expression of pobA, beginning at a position separated by 55 bp of DNA. The general mechanisms by which PobR exerts transcriptional control resemble those that typify the LysR family of transcriptional activators, a group from which PobR is evolutionarily remote.
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PMID:Regulation of p-hydroxybenzoate hydroxylase synthesis by PobR bound to an operator in Acinetobacter calcoaceticus. 802 Dec 13

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandem repeats of a heptapeptide with the consensus YSPTSPS. It has been shown that the heptapeptide repeat interacts directly with the general transcription factor TFIID. We report here that the CTD activates transcription when fused to the DNA-binding domain of GAL4. More importantly, we find that the proline-rich transcriptional activation domain of the CCAAT-box-binding factor CTF/NF1 contains a sequence with striking similarity to the heptapeptide repeats of the CTD. We show that this CTD-like motif is essential for the transcriptional activator function of the proline-rich domain of CTF/NF1. Deletion of and point mutations in this CTD-like motif abolish the transcriptional activator function of the proline-rich domain, while natural CTD repeats from RNA polymerase II are fully functional in place of the CTD-like motif. We further show that the proline-rich activation domain of CTF/NF1 interacts directly with the TATA-box-binding protein (TBP), and that a mutation in the CTD-like motif that abolishes transcriptional activation reduces the affinity of the proline-rich domain for TBP. These results demonstrate that a class of proline-rich activator proteins and RNA polymerase II possess a common structural and functional component which can interact with the same target in the general transcription machinery. We discuss the implications of these results for the mechanisms of transcriptional activation in eucaryotes.
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PMID:The upstream activator CTF/NF1 and RNA polymerase II share a common element involved in transcriptional activation. 802 1

A genetic response of Escherichia coli to nitric oxide or to superoxide-generating agents such as paraquat is controlled by the soxRS locus. The intracellular redox signals generated by these agents are sensed by the SoxR protein which, when activated, functions as a potent activator of soxS transcription. The resulting increased level of SoxS protein then activates approximately 10 genes that constitute the soxRS regulon. Although the SoxS protein is homologous to the COOH-terminal region of the AraC family of regulatory proteins, the mechanism by which SoxS protein activates the soxRS regulon promoters is unknown. We identified in extracts of cells expressing high levels of SoxS protein a DNA binding activity specific for fragments containing soxRS-regulated promoters. This binding activity was purified to physical homogeneity and proved to be the SoxS protein, as confirmed by NH2-terminal amino acid sequencing. The purified SoxS protein bound specifically to the promoters of the micF, zwf, nfo, and sodA genes. Multiple DNA-protein complexes were formed by SoxS in a concentration-dependent fashion with each of these promoters. This binding of SoxS protein also facilitated the subsequent binding of E. coli RNA polymerase to both the micF and the nfo promoters. The binding sites of SoxS in the zwf and micF promoters were identified by DNase I footprinting, which revealed an extended protected region immediately upstream of the respective -35 sites. These results indicate that the small SoxS protein (M(r) of only 12,900) is a direct transcriptional activator of the oxidative stress genes of the soxRS regulon, although the possible involvement of other proteins in transcription activation by SoxS has not been ruled out.
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PMID:SoxS, an activator of superoxide stress genes in Escherichia coli. Purification and interaction with DNA. 803 83

The core promoter of the human DNA beta-polymerase (beta-pol) gene is regulated by proteins binding at 3 GC boxes and the single activating transcription factor/cAMP response element (ATF/CRE) centered at -45; the central 8 residues of this ATF/CRE match the ATF/CRE consensus sequence, TGACGTCA. Previously, we purified a beta-pol promoter ATF/CRE-binding protein (named palindrome-binding protein or PBP) from bovine testes and found that this protein is a beta-pol promoter transcriptional activator in vitro using a HeLa nuclear extract transcription system (Widen, S. G., and Wilson, S. H. (1991) Biochemistry 30, 6296-6305). In this study, we determined the mechanism of in vitro transcriptional activation by this purified PBP. We used a PBP-depleted HeLa nuclear extract transcription system with an artificial promoter containing a solitary activator element corresponding to the entire 22-nucleotide beta-pol promoter ATF/CRE-binding site. Kinetic analyses of the 180-nucleotide run-off product formation indicated that stimulation of transcriptional activity by PBP was due entirely to an increase in the rate constant for promoter clearance. Thus, under our conditions, the purified PBP had no effect on the rate of closed preinitiation complex formation or for the closed complex to open complex transition. Instead, the rate of productive initiation leading to the 180-nucleotide transcript was stimulated by PBP. We found that the rate of closed preinitiation complex formation was not in rapid equilibrium with promoter and RNA polymerase II, in contrast to the model with prokaryotic RNA polymerase transcription. The results also indicated that PBP binding to the ATF/CRE is required for the stimulation of promoter clearance. These studies define the kinetic mechanism of a purified ATF/CRE-binding protein in stimulation of the in vitro transcription of a designed mammalian promoter.
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PMID:RNA polymerase II transcription. Rate of promoter clearance is enhanced by a purified activating transcription factor/cAMP response element-binding protein. 817 88


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