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 poliovirus RNA-dependent RNA polymerase required an oligouridylate primer or a HeLa cell protein (host factor) to initiate RNA synthesis on poliovirion RNA in vitro. The polymerase synthesized template-sized product RNA in the oligouridylate-primed reaction. In the host factor-dependent reaction, the largest product RNA synthesized by the polymerase was twice the size of the template RNA. About half of the product RNA recovered from this reaction was shown to exist in the form of a snapback sequence. Time-course reactions and pulse-chase experiments showed that the product RNA was only slightly larger than the template RNA at early reaction times and that with time it increased in size to form the dimer-sized product RNA. Inhibition of the elongation reaction by adding only [alpha-32P]UTP and ATP resulted in the formation of template-sized product RNA. The dimer-sized product RNA was unaffected by phenol extraction or proteinase K treatment but was converted to template-sized molecules by S1 nuclease. Dimer-sized poliovirus RNA that was sensitive to S1 nuclease was also isolated from poliovirus-infected cells. The results from this study indicate that the labeled negative-strand product RNA synthesized in vitro was covalently linked to the positive-strand template RNA. Thus, in vitro, the primer-dependent poliovirus RNA polymerase may initiate RNA synthesis in the presence of the host factor by using the 3' end of the template RNA as a primer.
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PMID:Poliovirus RNA-dependent RNA polymerase and host cell protein synthesize product RNA twice the size of poliovirion RNA in vitro. 298 94

Treatment of insect polyribosomes with 1 M KCl released a messenger ribonucleoprotein with a pronounced 16S peak. Phenol extraction resulted in a defined peak of 10S RNA, which was judged as mRNA by the following criteria: it showed specificity for binding to ribosomes, and the formation of initiation complex was dependent on protein initiation factors, GTP, mRNA, and aminoacyl-tRNA. The complex directed protein synthesis upon the addition of elongation factors. mRNA was treated with phosphatase and phosphorylated at the 5'-end with [(32)P]cyanoethylphosphate. [(32)P]mRNA was digested by T1 ribonuclease to completion and chromatographed on DEAE-cellulose. The only fragment with (32)P was 15 nucleotides long; it was treated with pancreatic ribonuclease and fingerprinted. Fractions of AC, AAC, and AAAC were found. Initiation signal AUG or GUG in these mRNAs does not begin immediately at the 5'-end and may be at a distance greater than 15 nucleotides. Alkaline hydrolysis of mRNAs labeled in vivo with [(14)C]adenosine revealed Ap and pppAp. Alkaline hydrolysis of mRNA labeled with (32)P at the 5'-terminus resulted in pAp. Hence, these results suggest that in a heterogeneous population of mRNAs from insects, all start with A and have sequence homology at the 5'-termini. This sequence may reflect the signal for RNA polymerase on the gene or may promote the binding of mRNA to ribosomes.
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PMID:Sequence homology at the 5'-termini of insect messenger RNAs. 435 Nov 73

The poly (A)-mRNA fraction isolated by chloroform deproteinization of liver polysomes and poly(U)-Sepharose chromatography contains a low molecular weights (congruent to 1000) peptidic fraction. The peptides which we suggested to call deprimerones (1) were extracted with 80% ethanol at pH 9.5; after ethanol evaporation, they were purified on Sephadex G-25 column as a fraction of mol. wt. between 1600 and 600, yielding about 9 mg/mg mRNA. If deproteinization is performed with phenol-chloroform the yield is about 2 mg/mg mRNA. In Novikoff hepatoma the yield of the same preparation is only 2.7 mg/mg mRNA (about 70% decrease). The obtained deprimerones are active in inhibiting transcription of thymus DNA with E. coli RNA polymerase and [3H]-GTP by about 90% at a ratio peptide/DNA = 2. For comparison the deprimerones obtained previously (2) by extraction of deproteinized DNA inhibit transcription only by about 50% at the same peptide/DNA ratio. The results demonstrated a decrease of the poly (A)-mRNA deprimerone level during carcinogenesis and further support the previously demonstrated specific occurrence of deprimerones with poly(A)-mRNA. They remain in accordance with and provide further support for the deprimerone theory of carcinogenesis postulated earlier (1).
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PMID:Poly(A)-mRNA deprimerones in rat liver and Novikoff hepatoma cells. 610 57

DNA isolated from rat liver by intensive deproteinization with chloroform/isoamyl alcohol and phenol contains low molecular weight peptides in a quantity of about 20 micrograms/mg DNA. These peptides show high specific activity in inhibiting transcription in a reconstituted cell-free system with prokaryotic and eukaryotic RNA polymerase. Their level is markedly decreased in DNA prepared from Novikoff hepatoma cells. Moreover the amino acid analysis and the pattern of analytical separation by high performance liquid chromatography (HPLC) show some biochemical differences between DNA-binding peptides extracted from rat liver and Novikoff hepatoma cells. The possibility that carcinogenesis may involve mechanisms which lead to selective removal of some components of the DNA-binding peptides, is discussed.
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PMID:DNA-binding peptides from rat liver and Novikoff hepatoma cells: quantitative level and possible biochemical differences. 668 24

DNA isolated from cell nuclei by intensive deproteinization with chloroform/isoamyl alcohol and phenol extractons contains a low molecular weight peptidic fraction in a quantity of about 20 micrograms/mg DNA. These peptides were characterized by chromatography on CM-Sephadex, Sephadex G-25, high performance liquid chromatography on microBondapak C18 and amino acid composition. The peptides control transcription in a reconstituted cell-free system with prokaryotic and eukaryotic RNA polymerase and stabilize the structure of double stranded DNA, while increasing its melting point. Their level is markedly decreased (by about 40%) in DNA prepared from tumor cells as compared to normal cell DNA. Transcriptional studies showed only a slightly increased template activity of DNA extracted at pH 9.5 versus DNA extracted at pH 6.0 for DNA preparations from tumor cells. However, there was a marked increase in template activity for DNA preparations treated at pH 9.5 from normal cells--232%, 124%, 97% and 78% for rat liver, mouse liver, mouse thymus and fibroblast L-929 cells, respectively. Also there was no difference in the melting point between these two preparations of DNA from tumor cells; normal cell DNA preparations showed increased melting point of preparations treated at pH 6.5. The data obtained indicate that the loss of low molecular weight peptides from tumor DNA during carcinogenesis is responsible for uncontrolled gene expression observed in cancer.
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PMID:Low molecular weight peptidic fraction in the chromatin from normal and cancer cells: control of transcription. 741 71

A pathogenicity locus in Xanthomonas campestris pv. campestris has been shown to comprise two genes which mediate biosynthesis of the bacterial lipopolysaccharide (LPS) but not extracellular polysaccharide. Mutants with Tn5 insertions in either gene showed alterations in the electrophoretic patterns of both water-soluble and phenol-soluble LPS forms, which suggested defects in the biosynthesis of the core oligosaccharide component. On gel chromatography, core oligosaccharides of the mutants were of apparently lower molecular weight than those from the wild type. Furthermore, the content of mannose and glucose, sugars characteristic of the core oligosaccharide, were significantly lower in the water-soluble LPS of the mutants. Because of their role in LPS core biosynthesis, the two genes were called rfaX and rfaY. rfaX mutants show altered behavior in a range of host and non-host plants such that the number of recoverable bacteria drop within the first 24 h after inoculation. In contrast, the behavior of rfaY mutants only differed from the wild type in Datura, a non-host plant in which the growth of the wild type is severely attenuated. The predicted protein RfaY showed significant sequence homology to a sub-family of RNA polymerase sigma factors which are involved in extracytoplasmic functions.
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PMID:A locus determining pathogenicity of Xanthomonas campestris is involved in lipopolysaccharide biosynthesis. 757 21

Pseudomonas putida P35X (NCIB 9869) metabolizes phenol and monomethylphenols via a chromosomally encoded meta-cleavage pathway. We have recently described a 13.4-kb fragment of the chromosome that codes for the first eight genes of the catabolic pathway and a divergently transcribed positive regulator, phhR. The eight structural genes lie in an operon, the phh operon, downstream of a -24 TGGC, -12 TTGC promoter sequence. Promoters of this class are recognized by RNA polymerase that utilizes the alternative sigma 54 factor encoded by rpoN (ntrA) and are positively regulated by activators of the NtrC family. In this study, we have identified the coding region for the 63-kDa PhhR gene product by nucleotide sequencing of a 2,040-bp region and polypeptide analysis. PhhR was found to have homology with the NtrC family of transcriptional activators, in particular with DmpR, the pVI150-encoded regulator of (methyl)phenol catabolism by Pseudomonas sp. strain CF600. By using a luciferase reporter system, PhhR alone was shown to be sufficient to activate transcription from the phh operon promoter in an RpoN+ background but not an RpoN- background. Luciferase reporter systems were also used to directly compare the aromatic effector profiles of PhhR and DmpR. Evidence that the difference in the growth substrate ranges of strains P35X and CF600 is due to the effector activation specificities of the regulators of these systems rather than the substrate specificities of the catabolic enzymes is presented.
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PMID:Aromatic effector activation of the NtrC-like transcriptional regulator PhhR limits the catabolic potential of the (methyl)phenol degradative pathway it controls. 788 4

Plasmid pEST1463 carrying the promoterless pheBA operon was cloned into Pseudomonas putida PaW85, and phenol-utilizing colonies were isolated on minimal plates containing phenol as the only carbon and energy source. In these clones, chromosomally located Tn4652 was transposed upstream from the coding sequencing of pheA (encoding phenol monooxygenase). Sequence analysis together with mapping of the transcription start point of the pheBA operon in the recombinant plasmids revealed that fusions of the -10 sequences present in the pheBA operon and -35 sequence located in the terminal inverted repeats of Tn4652 had generated functional promoters under selective pressure in P. putida cells. These promoter sequences show similarity to the Escherichia coli RNA polymerase sigma 70 promoter consensus sequence. In three of the six fusion promoters studied, the generation combined two distinct events: transposition of Tn4652 into DNA containing potential -10 sequences and point mutations in these sequences. These mutations made the -10 sequences more like the sigma 70 promoter consensus sequences.
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PMID:In-vivo-generated fusion promoters in Pseudomonas putida. 838 46

A method was developed for fast and efficient isolation of RNA from paraffin-embedded tissue sections for subsequent PCR analysis. This method is based on the binding of RNA to acid-treated glass beads in the presence of a high molarity of guanidinium salt. It can be completed within an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was tested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the extracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. PCR amplification of cellular and viral RNA was successful for RNA isolated by use of all extraction techniques, although the glass bead method was preferred for its simplicity and rapidity. Specimens fixed with formalin were found to be suitable for PCR, but the best results were obtained with acetone-fixed paraffin-embedded material. Dewaxing of tissue sections had no effect on the yield and quality of RNA extractions, and further purification of the extracts using gel filtration did not improve the results. After the protocols were optimized, rotavirus-infected cell pellets were used to demonstrate that extraction and amplification of dsRNA was possible. The information obtained from the studies with the model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin-embedded archival tissue can be used for RT-PCR analysis, adding an important technique to diagnostic pathology and retrospective studies.
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PMID:Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives. 839 55

The catabolic plasmid pVI150 of Pseudomonas sp. strain CF600 encodes all the genetic information required for the regulated metabolism of phenol and some of its methyl-substituted derivatives. The structural dmp genes of the pathway are clustered in a single operon that lies just downstream of a -24 TGGC, -12 TTGC nif/ntr-like promoter sequence. Promoters of this class are recognized by a minor form of RNA polymerase utilizing sigma 54 (NtrA, RpoN). Primer extension analysis demonstrated that the dmp operon transcript initiates downstream of the -24, -12 promoter. Transposon insertion mutants, specifically defective in the regulation of the dmp operon, were isolated, and complementation of a phenol-utilization regulatory mutant was used to identify the regulatory locus, dmpR. The 67-kDa dmpR gene product alone was shown to be sufficient for activation of transcription from the dmp operon promoter. Nucleotide sequence determination revealed that DmpR belongs to the NtrC family of transcriptional activators that regulate transcription from -24, -12 promoters. The deduced amino acid sequence of DmpR has high homology (40 to 67% identity) with the central and carboxy-terminal regions of these activators, which are believed to be involved in the interaction with the sigma 54 RNA polymerase and in DNA binding, respectively. The amino-terminal region of DmpR was found to share 64% identity with the amino-terminal region of XylR, which is also a member of this family of activators. This region has been implicated in effector recognition of aromatic compounds that is required for the regulatory activity of XylR.
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PMID:Cloning and nucleotide sequence of the gene encoding the positive regulator (DmpR) of the phenol catabolic pathway encoded by pVI150 and identification of DmpR as a member of the NtrC family of transcriptional activators. 844 69


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