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 used a sequence-specific DNA binding protein to examine transcription elongation and termination by mammalian RNA polymerase III (polIII). The Escherichia coli lac repressor protein, bound to its cognate operator site positioned between the 3' end of the coding region and the termination site of a human tRNA gene, conditionally blocked transcription elongation by polIII in vitro in HeLa cell nuclear extracts. Arrest of elongation by polIII dramatically reduced overall levels of transcription and directed the synthesis of shortened transcripts, consistent with a block to polIII elongation at the boundary of the repressor/DNA complex. Removal of template-bound repressor with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG) allowed extension of nascent transcripts and restored transcriptional activity. Moreover, a subset of transcription complexes were shown to be capable of transcribing through the repressor obstacle. lac repressor positioned just downstream of the natural termination site effected the premature termination of transcription but otherwise had no affect on the overall level of transcription. Our findings demonstrate that elongation and termination by mammalian polIII can be modulated in vitro by a heterologous sequence-specific DNA binding protein. Moreover, the ability to selectivity arrest elongation by polIII at defined positions within the tRNA gene transcription unit has permitted the identification of discrete functional properties of paused mammalian polIII ternary complexes.
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PMID:RNA chain elongation and termination by mammalian RNA polymerase III. Analysis of tRNA gene transcription by imposing a reversible factor-mediated block to elongation using a sequence-specific DNA binding protein. 799 Jan 36

Saccharomyces cerevisiae transcription factor IIIA, a sequence-specific DNA binding protein that is required for transcription of 5S rRNA genes by RNA polymerase III, has been expressed in Escherichia coli in a full length, native form. High level expression was achieved through the combined use of a T7 RNA polymerase expression system and of a multicopy plasmid carrying an E. coli gene, argU, which codes for a minor Arg(AGA/AGG) tRNA species. Recombinant yeast transcription factor IIIA was purified to 95% homogeneity, at a final yield of 8 mg/liter of bacterial culture, by three chromatographic steps, and it was shown to be at least 55% active by quantitative in vitro transcription assays.
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PMID:High level expression in E. coli and purification of yeast transcription factor IIIA. 809 41

In Drosophila and human cells, the TATA binding protein (TBP) of the transcription factor IID (TFIID) complex is tightly associated with multiple subunits termed TBP-associated factors (TAFs) that are essential for mediating regulation of RNA polymerase II transcription. The Drosophila TAFII150 has now been molecularly cloned and biochemically characterized. The deduced primary amino acid sequence of dTAFII150 reveals a striking similarity to the essential yeast gene, TSM-1. Furthermore, like dTAFII150, the TSM-1 protein is found associated with the TBP in vivo, thus identifying the first yeast homolog of a TAF associated with TFIID. Both the product of TSM-1 and dTAFII150 bind directly to TBP and dTAFII250, demonstrating a functional similarity between human and yeast TAFs. Surprisingly, DNA binding studies indicate that purified recombinant dTAFII150 binds specifically to DNA sequences overlapping the start site of transcription. The data demonstrate that at least one of the TAFs is a sequence-specific DNA binding protein and that dTAFII150 together with TBP are responsible for TFIID interactions with an extended region of the core promoter.
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PMID:Drosophila TAFII150: similarity to yeast gene TSM-1 and specific binding to core promoter DNA. 817 53

We have previously characterized a protein, enhancer 1 binding factor (E1BF), from rat cells that can modulate RNA polymerase I-directed transcription of the rat rRNA gene in vitro. E1BF, a heterodimeric DNA binding protein composed of 72-kDa and 85-kDa subunits, is related to the human Ku autoantigen with respect to immunological and certain structural properties. To establish the direct role of E1BF in transcription, we investigated the effect of anti-Ku antibodies on RNA polymerase I-directed transcription in rat and mouse cell extracts. These antibodies, one directed against the 70-kDa Ku subunit and the other against a peptide fragment of this subunit, dissociated the E1BF heterodimer into its two subunits. The DNA-protein complex formed in the presence of the antibodies contained only the 72-kDa subunit. Preincubation of the extracts with these antibodies resulted in an almost complete inhibition of transcription. The reduced transcription was observed when either linear or circular template was used. The inhibitory effect of the antibodies was greatest when added prior to preinitiation complex formation and was minimized significantly when added after establishment of the initiation complex. The repression of rRNA gene transcription was overcome by the addition of purified E1BF. This study demonstrates that E1BF, a Ku-related protein, is required for RNA polymerase I-directed transcription, the 72-kDa subunit is the major DNA binding polypeptide, the factor acts primarily in the formation of the preinitiation complex, and heterodimerization of its two subunits is crucial for maintaining the functional integrity of the protein.
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PMID:Enhancer 1 binding factor, a Ku-related protein, is a positive regulator of RNA polymerase I transcription initiation. 829 May 97

Inactivation of Bacillus subtilis orf1177 in an otherwise Rec+ strain reduced genetic exchange and DNA repair. When the mutation was transferred into a set of recombination-deficient and repair-deficient strains, the DNA repair and recombination ability of the double or triple mutant strains was drastically reduced. B. subtilis Orf1177 protein shares substantial homology with the Escherichia coli Mdf, RecG and UvrB proteins. In vivo analysis of UV-induced mutations suggests that Orf1177 is necessary for strand-specific DNA repair, as is the case for the E. coli MFD protein. Therefore, orf1177 and Orf1177 were termed mfd gene and Mfd protein, respectively. The purified Mfd protein has a native molecular mass of 140 kDa (expected molecular mass 133 kDa). The Mfd protein is a sequence-independent DNA binding protein with weak ATPase activity. The Mfd protein was able to displace in vitro B. subtilis or E. coli RNA polymerase stalled at a lesion. Therefore, Mfd protein appears to target the transcribed strand for repair by recognizing a stalled RNA polymerase and dissociating it from the DNA. In addition, the strong recombination-deficient phenotype of mfd- rec- strains suggest that Mfd protein is involved in homologous DNA recombination.
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PMID:The Mfd protein of Bacillus subtilis 168 is involved in both transcription-coupled DNA repair and DNA recombination. 859 98

A Z-DNA binding protein of 140,000 M(r) has been purified from chicken lungs by sedimentation through 40%(w/w) sucrose and Z-DNA affinity chromatography. Specificity of the protein for Z-DNA was confirmed by competition with polyd(CG) that had been stabilized in the Z-DNA conformer by chemical bromination and also with a supercoiled plasmid that contains a Z-DNA-forming insert. In addition to a Z-DNA binding site, the protein also has a separate binding site for double-stranded RNA. Peptide sequence of the protein shows that it has high similarity to the RNA editing enzyme double-stranded RNA adenosine deaminase (dsRAD), which deaminates adenosine in dsRNA to form inosine. The Z-DNA binding protein has this enzymatic activity, confirming its identity to dsRAD. Recombinant human dsRAD also binds to Z-DNA. Z-DNA is stabilized in a sequence-dependent manner by negative supercoiling, which occurs in actively transcribed genes upstream to RNA polymerase. It is proposed that Z-DNA links editing to transcription by localizing dsRAD to a particular region of a gene and thus determines the efficiency with which an RNA is edited. The presence of Z-DNA forming elements in many genes raises the possibility that RNA editing by dsRAD is far more prevalent than is currently thought.
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PMID:Double-stranded RNA adenosine deaminase binds Z-DNA in vitro. 864 57

Although the majority of the transcription factors that were initially characterized had a stimulatory effect on gene expression, it is now clear that transcription factors that inhibit gene transcription are at least as important in regulating a wide variety of processes, including development. These factors can act either by interfering with the action of a positively acting factor (indirect repression) or by directly interfering with transcription by interacting with the basal transcriptional complex of RNA polymerase and associated factors (direct repression). Indirect repression often operates by the negative factor preventing the positively acting factor binding to DNA. This can involve reorganization of chromatin structure, blockage of the binding site in the DNA by binding of the inhibitory factor or formation of a non-DNA binding protein-protein complex. Indirect repression can also occur via quenching of the activity of a positive factor that remains bound to DNA. Direct repression can be produced by factors that interact with the basal transcriptional complex to reduce its activity or stability. This can be achieved either by factors that interact with the complex following binding to DNA or by those which bind directly to it. Factors that act by each of these means, and their mechanisms of action, are discussed.
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PMID:Inhibitory transcription factors. 893 Jan 19

The coding sequences for a Schizosaccharomyces pombe sequence-specific DNA binding protein, Reb1p, have been cloned. The predicted S. pombe Reb1p is 24-29% identical to mouse TTF-1 (transcription termination factor-1) and Saccharomyces cerevisiae REB1 protein, both of which direct termination of RNA polymerase I catalyzed transcripts. The S.pombe Reb1 cDNA encodes a predicted polypeptide of 504 amino acids with a predicted molecular weight of 58.4 kDa. The S. pombe Reb1p is unusual in that the bipartite DNA binding motif identified originally in S.cerevisiae and Klyveromyces lactis REB1 proteins is uninterrupted and thus S.pombe Reb1p may contain the smallest natural REB1 homologous DNA binding domain. Its genomic coding sequences were shown to be interrupted by two introns. A recombinant histidine-tagged Reb1 protein bearing the rDNA binding domain has two homologous, sequence-specific binding sites in the S. pomber DNA intergenic spacer, located between 289 and 480 nt downstream of the end of the approximately 25S rRNA coding sequences. Each binding site is 13-14 bp downstream of two of the three proposed in vivo termination sites. The core of this 17 bp site, AGGTAAGGGTAATGCAC, is specifically protected by Reb1p in footprinting analysis.
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PMID:Molecular cloning and analysis of Schizosaccharomyces pombe Reb1p: sequence-specific recognition of two sites in the far upstream rDNA intergenic spacer. 901 45

We have previously demonstrated that the core promoter of rat ribosomal RNA gene (rDNA) contains an E-box-like sequence to which the core promoter binding factor CPBF binds and that the 44 kDa subunit of this protein is immunologically related to USF1, the helix--loop--helix-zipper DNA binding protein. Further, we showed that RNA polymerase I (pol I) transcription in vitro is competed by oligonucleotides containing USF-binding site, which suggested a key role for USF in rDNA transcription. To prove the potential role of USF in pol I transcription in vivo, USF1 and USF2 homodimers and USF1/USF2 heterodimer were overexpressed in CHO cells by transfection of the respective cDNAs. Co-transfection of a plasmid containing rDNA followed by primer extension analysis showed that overexpression of USF1 and USF2 as homodimers resulted in inhibition of rDNA transcription by as much as 85-90% whereas overexpression of USF1/USF2 in the heterodimeric form activated transcription approximately 3.5-fold. Transfection of mutant USF2 cDNA that is devoid of the basic DNA-binding domain produced only minimal inhibition of rDNA transcription. These data show that USF can modulate transcription of rRNA gene in vivo by functioning as a repressor (homodimer) or activator (heterodimer) of pol I transcription in vivo and suggest that inhibition of rDNA transcription may be responsible for the antiproliferative action of USF homodimers.
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PMID:The dual role of helix-loop--helix-zipper protein USF in ribosomal RNA gene transcription in vivo. 905 57

Yeast Cbf5p was originally isolated as a low-affinity centromeric DNA binding protein (W. Jiang, K. Middleton, H.-J. Yoon, C. Fouquet, and J. Carbon, Mol. Cell. Biol. 13:4884-4893, 1993). Cbf5p also binds microtubules in vitro and interacts genetically with two known centromere-related protein genes (NDC10/CBF2 and MCK1). However, Cbf5p was found to be nucleolar and is highly homologous to the rat nucleolar protein NAP57, which coimmunoprecipitates with Nopp140 and which is postulated to be involved in nucleolar-cytoplasmic shuttling (U. T. Meier, and G. Blobel, J. Cell Biol. 127:1505-1514, 1994). The temperature-sensitive cbf5-1 mutant demonstrates a pronounced defect in rRNA biosynthesis at restrictive temperatures, while tRNA transcription and pre-rRNA and pre-tRNA cleavage processing appear normal. The cbf5-1 mutant cells are deficient in cytoplasmic ribosomal subunits at both permissive and restrictive temperatures. A high-copy-number yeast genomic library was screened for genes that suppress the cbf5-1 temperature-sensitive growth phenotype. SYC1 (suppressor of yeast cbf5-1) was identified as a multicopy suppressor of cbf5-1 and subsequently was found to be identical to RRN3, an RNA polymerase I transcription factor. A cbf5delta null mutant is not rescued by plasmid pNOY103 containing a yeast 35S rRNA gene under the control of a Pol II promoter, indicating that Cbf5p has one or more essential functions in addition to its role in rRNA transcription.
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PMID:The yeast nucleolar protein Cbf5p is involved in rRNA biosynthesis and interacts genetically with the RNA polymerase I transcription factor RRN3. 931 78


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