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 isolated and analyzed human CTCF cDNA clones and show here that the ubiquitously expressed 11-zinc-finger factor CTCF is an exceptionally highly conserved protein displaying 93% identity between avian and human amino acid sequences. It binds specifically to regulatory sequences in the promoter-proximal regions of chicken, mouse, and human c-myc oncogenes. CTCF contains two transcription repressor domains transferable to a heterologous DNA binding domain. One CTCF binding site, conserved in mouse and human c-myc genes, is found immediately downstream of the major P2 promoter at a sequence which maps precisely within the region of RNA polymerase II pausing and release. Gel shift assays of nuclear extracts from mouse and human cells show that CTCF is the predominant factor binding to this sequence. Mutational analysis of the P2-proximal CTCF binding site and transient-cotransfection experiments demonstrate that CTCF is a transcriptional repressor of the human c-myc gene. Although there is 100% sequence identity in the DNA binding domains of the avian and human CTCF proteins, the regulatory sequences recognized by CTCF in chicken and human c-myc promoters are clearly diverged. Mutating the contact nucleotides confirms that CTCF binding to the human c-myc P2 promoter requires a number of unique contact DNA bases that are absent in the chicken c-myc CTCF binding site. Moreover, proteolytic-protection assays indicate that several more CTCF Zn fingers are involved in contacting the human CTCF binding site than the chicken site. Gel shift assays utilizing successively deleted Zn finger domains indicate that CTCF Zn fingers 2 to 7 are involved in binding to the chicken c-myc promoter, while fingers 3 to 11 mediate CTCF binding to the human promoter. This flexibility in Zn finger usage reveals CTCF to be a unique "multivalent" transcriptional factor and provides the first feasible explanation of how certain homologous genes (i.e., c-myc) of different vertebrate species are regulated by the same factor and maintain similar expression patterns despite significant promoter sequence divergence.
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PMID:An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes. 864 89

ohrR encodes a novel organic peroxide-inducible transcription repressor, and we have demonstrated that ohrR is regulated at the transcriptional and the post-transcriptional levels. Primer extension results show that ohrR transcription initiates at the A residue of the ATG translation initiation codon for the ohrR coding sequence. Thus, the gene has a leaderless mRNA. The ohrR promoter (P1) has high homology to the consensus sequence for Xanthomonas promoters, which is reflected in the high in vivo promoter activity of P1. Deletion of a 139 bp fragment containing the P1 promoter showed that the sequences upstream of -35 regions were required for neither the promoter activity nor OhrR autoregulation. In vitro, purified OhrR specifically binds to the P1 promoter. DNase I footprinting of OhrR binding to the P1 revealed a 44 bp region of protection on both DNA strands. The protected regions include the -35 and -10 regions of P1. We suggest that OhrR represses gene expression by blocking RNA polymerase binding to the promoter. There are two steps in the post-transcriptional regulation of ohrR, namely differential stability and inefficient translation of the mRNA. The bicistronic ohrR-ohr mRNA was highly labile and underwent rapid processing in vivo to give only stable monocistronic ohr mRNA and undetectable ohrR mRNA. Furthermore, the ohrR mRNA was inefficiently translated. We propose that, in uninduced cells, the concentration of OhrR is maintained at low levels by the autoregulation mechanism at the transcriptional levels and by the ohrR mRNA instability coupled with inefficient translation at the post-transcriptional level. Upon exposure to an organic peroxide, the compound probably interacts with OhrR and prevents it from repressing the P1 promoter, thus allowing high-level expression of the ohrR-ohr operon. The rapid processing of bicistronic mRNA gives highly stable ohr mRNA and corresponding high levels of Ohr, which remove an organic per-oxide. Once the peroxide has been removed, the autoregulation mechanism feeds back to inhibit the expression of the operon.
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PMID:The repressor for an organic peroxide-inducible operon is uniquely regulated at multiple levels. 1199 59

The EVI1 proto-oncogene encodes a nuclear zinc finger protein that acts as a transcription repressor factor. In myeloid leukemia it is often activated by chromosomal rearrangements involving band 3q26, where the gene has been mapped. Here we report two leukemia cases [a chronic myeloid leukemia blast crisis (CML-BC) and an acute myeloid leukemia (AML) M4] showing a t(3;7)(q26;q21) translocation in a balanced and unbalanced form, respectively. Fluorescent in situ hybridization (FISH) analysis revealed that both patients showed a breakpoint on chromosome 3 inside the clone RP11-33A1 containing the EVI1 oncogene and, on chromosome 7, inside the clone RP11-322M5, partially containing the CDK6 oncogene which is a D cyclin-dependent kinase gene, observed to be overexpressed and disrupted in many hematological malignancies. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed overexpression of EVI1 in both cases, but excluded the presence of any CDK6/ EVI1 fusion transcript. CDK6 expression was also detected. Together, these data indicate that EVI1 activation is likely due not to the generation of a novel fusion gene with CDK6 but to a position effect dysregulating its transcriptional pattern.
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PMID:A novel chromosomal translocation t(3;7)(q26;q21) in myeloid leukemia resulting in overexpression of EVI1. 1455 38

The oncogenic potential of latent Epstein-Barr virus (EBV) can be regulated by epigenetic factors controlling LMP1 and EBNA2 gene transcription. The EBV latency control region (LCR) constitutes approximately 12 kb of viral sequence spanning the divergent promoters of LMP1 and EBNA2 and encompasses the EBV latent replication origin OriP and RNA polymerase III-transcribed EBV-encoded RNA genes. We have used the chromatin immunoprecipitation assay to examine the chromatin architecture of the LCR in different types of EBV latency programs. We have found that histone H3 K4 methylation (H3mK4) was enriched throughout a large domain that extended from internal repeat 1 (IR1) to the terminal repeat in type III latency where EBNA2 and LMP1 genes are expressed. In type I latency where EBNA2 and LMP1 genes are transcriptionally silent, the H3mK4 domain contracts and does not enter the EBNA2 or LMP1 promoters. In contrast, histone H3 K9 methylation (H3mK9), associated with silent heterochromatin, was enriched in the EBNA2 and LMP1 upstream control regions in type I but not type III cells. MTA [5'-deoxy-5'(methylthio)adenosine], a pharmacological inhibitor of protein methylation, globally reduced histone H3mK4 and inhibited EBNA2 transcription in type III cells. 5'-Azacytidine, an inhibitor of DNA methylation that derepresses EBNA2 transcription in type I latency, caused H3mK4 expansion and a corresponding loss of H3mK9 at IR1. The chromatin boundary protein and transcription repressor CCCTC-binding factor was enriched at the EBNA2 transcription control region in type I but not type III cells. We also present evidence that OriP binding factors EBNA1 and ORC2 can interact with sequences outside of OriP including a region within IR1 that may influence EBNA2 transcription status. These results indicate that types I and III latency programs have distinct histone methylation patterns in the LCR and suggest that chromatin architecture coordinates gene expression of LMP1 and EBNA2.
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PMID:Dynamic chromatin boundaries delineate a latency control region of Epstein-Barr virus. 1550 18

Human T-lymphotropic virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose function is essential for viral transcription and replication. Tax transactivates the viral long-terminal repeat through a series of protein-protein interactions which facilitate CREB and CBP/p300 binding. In addition, Tax dissociates transcription repressor histone deacetylase 1 interaction with the CREB response element. The subsequent events through which Tax interacts and communicates with RNA polymerase II and cyclin-dependent kinases (CDKs) are not clearly understood. Here we present evidence that Tax recruits positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T1) to the viral promoter. This recruitment likely involves protein-protein interactions since Tax associates with P-TEFb in vitro as demonstrated by glutathione S-transferase fusion protein pull-down assays and in vivo as shown by co-immunoprecipitation assays. Functionally, small interfering RNA directed toward CDK9 inhibited Tax transactivation in transient assays. Consistent with these findings, the depletion of CDK9 from nuclear extracts inhibited Tax transactivation in vitro. Reconstitution of the reaction with wild-type P-TEFb, but not a kinase-dead mutant, recovered HTLV-1 transcription. Moreover, the addition of the CDK9 inhibitor flavopiridol blocked Tax transactivation in vitro and in vivo. Interestingly, we found that Tax regulates CDK9 kinase activity through a novel autophosphorylation pathway.
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PMID:Tax interacts with P-TEFb in a novel manner to stimulate human T-lymphotropic virus type 1 transcription. 1664 Dec 71

Protein-DNA complexes must be disassembled to facilitate DNA replication. Replication forks contain a helicase that unwinds the duplex DNA at the front of the fork. The minichromosome maintenance helicase from the archaeon Methanothermobacter thermautotrophicus required only ATP to unwind DNA bound into complexes by the M. thermautotrophicus archaeal histone HMtA2, transcription repressor TrpY, or into a transcription pre-initiation complex by M. thermautotrophicus TATA-box-binding protein, transcription factor B, and RNA polymerase. In contrast, the minichromosome maintenance helicase was unable to unwind DNA bound by this archaeal RNA polymerase in a stalled transcript-elongating complex.
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PMID:Archaeal minichromosome maintenance (MCM) helicase can unwind DNA bound by archaeal histones and transcription factors. 1715 92

RNA polymerase III (Pol III) produces essential components of the biosynthetic machinery, and therefore its activity is tightly coupled with cell growth and metabolism. In the yeast Saccharomyces cerevisiae, Maf1 is the only known global and direct Pol III transcription repressor which mediates numerous stress signals. Here we demonstrate that transcription regulation by Maf1 is not limited to stress but is important for the switch between fermentation and respiration. Under respiratory conditions, Maf1 is activated by dephosphorylation and imported into the nucleus. The transition from a nonfermentable carbon source to that of glucose induces Maf1 phosphorylation and its relocation to the cytoplasm. The absence of Maf1-mediated control of tRNA synthesis impairs cell viability in nonfermentable carbon sources. The respiratory phenotype of maf1-Delta allowed genetic suppression studies to dissect the mechanism of Maf1 action on the Pol III transcription apparatus. Moreover, in cells grown in a nonfermentable carbon source, Maf1 regulates the levels of different tRNAs to various extents. The differences in regulation may contribute to the physiological role of Maf1.
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PMID:Maf1 is involved in coupling carbon metabolism to RNA polymerase III transcription. 1778 43

In mammals, NRF-2 (nuclear respiratory factor 2), also named GA-binding protein, is an Ets family transcription factor that controls many genes involved in cell cycle progression and protein synthesis as well as in mitochondrial biogenesis. In this paper, we analyzed the role of NRF-2 in the regulation of human genes involved in mitochondrial DNA transcription and replication. By a combination of bioinformatic and biochemical approaches, we found that the factor binds in vitro and in vivo to the proximal promoter region of the genes coding for the transcription termination factor mTERF, the RNA polymerase POLRMT, the B subunit of the DNA polymerase-gamma, the DNA helicase TWINKLE, and the single-stranded DNA-binding protein mtSSB. The role of NRF-2 in modulating the expression of those genes was further established by RNA interference and overexpression strategies. On the contrary, we found that NRF-2 does not control the genes for the subunit A of DNA polymerase-gamma and for the transcription repressor MTERF3; we suggest that these genes are under regulatory mechanisms that do not involve NRF proteins. Since NRFs are known to positively control the expression of transcription-activating proteins, the novelty emerging from our data is that proteins playing antithetical roles in mitochondrial DNA transcription, namely activators and repressors, are under different regulatory pathways. Finally, we developed a more stringent consensus with respect to the general consensus of NRF-2/GA-binding protein when searching for NRF-2 binding sites in the promoter of mitochondrial proteins.
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PMID:Nuclear respiratory factor 2 induces the expression of many but not all human proteins acting in mitochondrial DNA transcription and replication. 1995 46

NsrR is a nitric oxide (NO)-sensitive transcription repressor that controls NO metabolism in a wide range of bacteria. In Bacillus subtilis, NsrR represses transcription of the nitrite reductase (nasDEF) genes that are under positive control of the ResD-ResE two-component signal transduction system. Derepression is achieved by reaction of NO with NsrR. Unlike some NsrR orthologues that were shown to contain a NO-sensitive [2Fe-2S] cluster, B. subtilis NsrR, when purified anaerobically either from aerobic or from anaerobic Escherichia coli and B. subtilis cultures, contains a [4Fe-4S] cluster. [4Fe-4S]-NsrR binds around the -35 element of the nasD promoter with much higher affinity than apo-NsrR and binding of [4Fe-4S]-NsrR, but not apo-protein, is sensitive to NO. RNA polymerase and phosphorylated ResD make a ternary complex at the nasD promoter and NsrR dissociates the preformed ternary complex. In addition to the -35 region, NsrR binds to two distinct sites of the upstream regulatory region where ResD also binds. These interactions, unlike the high-affinity site binding, do not depend on the NsrR [4Fe-4S] cluster and binding is not sensitive to NO, suggesting a role for apo-NsrR in transcriptional regulation.
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PMID:Nitric oxide-sensitive and -insensitive interaction of Bacillus subtilis NsrR with a ResDE-controlled promoter. 2109 10

The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.
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PMID:Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. 2376 37


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