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 mechanism for elongation catalyzed by human RNA polymerase II (RNAP II) has been analyzed using millisecond phase transient state kinetics. Here, we apply a running start, two-bond, double-quench protocol. Quenching the reaction with EDTA indicates NTP loading into the active site followed by rapid isomerization. HCl quenching defines the time of phosphodiester bond formation. Model-independent and global kinetic analyses were applied to simulate the RNAP II mechanism for forward elongation through the synthesis of two specific phosphodiester bonds, modeling rate data collected over a wide range of nucleoside triphosphate concentrations. We report adequate two-bond kinetic simulations for the reaction in the presence of TFIIF alone and in the presence of TFIIF+TFIIS, providing detailed insight into the RNAP II mechanism and into processive RNA synthesis. RNAP II extends an RNA chain through a substrate induced-fit mechanism, termed NTP-driven translocation. After rapid isomerization, chemistry is delayed. At a stall point induced by withholding the next templated NTP, RNAP II fractionates into at least two active and one paused conformation, revealed as different forward rates of elongation. In the presence of TFIIF alone or in the presence of TFIIF+TFIIS, rapid rates are very similar; although, with TFIIF alone the complex is more highly poised for forward synthesis. Based on steady-state analysis, TFIIF was thought to suppress transcriptional pausing, but this view is misleading. TFIIF supports elongation and suppresses pausing by stabilizing the post-translocated elongation complex. When TFIIS is present, RNA cleavage and transcriptional restart pathways are supported, but TFIIS has a role in suppression of transient pausing, which is the most important contribution of TFIIS to elongation from a stall position.
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PMID:Transcription factors IIF and IIS and nucleoside triphosphate substrates as dynamic probes of the human RNA polymerase II mechanism. 1535 37

Previously, the lysozyme gene of the Klebsiella phage K11 was partially sequenced in our lab. Using the sequence information the lysozyme gene of the Klebsiella phage K11 was amplified and cloned using the polymerase chain reaction of the pfu DNA polymerase. The nucleotide sequence of phage K11 lysozyme gene was determined. The open reading frame corresponds to a polypeptide with 151 amino acids and molecular weight of 16,932 Da. The deduced amino acid sequence of this polypeptide shows 74-75% homologies to the T7 and T3 phage lysozymes. Although the gene was efficiently expressed under the control of tac promoter in Escherichia coli XL1-blue cells at 37 degrees C, most of the K11 lysozyme produced was insoluble. When the temperature of cell growth was lowered, however, solubility of the K11 lysozyme was increased gradually. The insoluble protein expressed at 37 degrees C was solubilized in 5 M guanidine-HCl and refolded in the presence of oxido-shuffling agent (GSH/GSSG). Through the refolding process the recombinant lysozyme was solubilized and purified. The purified K11 lysozyme showed transcription inhibition of K11 RNA polymerase as well as amidase activity. These results showed that the lysozyme of bacteriophage K11 is a bifunctional protein that cuts a bond in the bacterial cell wall and selectively inhibits K11 phage RNA polymerase. Also, transcription inhibition ability of K11 lysozyme with T7 or SP6 phage RNA polymerase was measured. T7 RNA polymerase was less inhibited than K11 RNA polymerase by K11 lysozyme. But SP6 RNA polymerase was not nearly inhibited by K11 lysozyme.
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PMID:Cloning and expression of Klebsiella phage K11 lysozyme gene. 1588 50

Facile evaluation of mixed-salt effect on the strongly salt-dependent thermodynamic and kinetic parameters of protein-DNA complexes is of importance for relevant biochemical and biophysical studies. In pursuit of this aim, binding isotherms for open transcription complex (RPo) of Escherichia coli RNA polymerase (R) at lambdaP(R) promoter DNA (P) were determined as a function of salt concentration in pure NaCl and Tris/HCl solutions, and as a function of [NaCl] in the presence of fixed concentrations of MgCl(2) and Tris/HCl. A concept of equivalent salt concentrations, i.e. concentrations at which the binding equilibrium constant is the same, was introduced and applied for prediction of binding isotherms in mixed salt solutions. Full coincidence between the experimental and predicted isotherms indicated that individual contributions of salts to the global salt-effect are additive in a broad range of salt concentrations. A generalized formula for calculation of salt equivalents characteristic for any of the thermodynamic or kinetic parameters of a complex (e.g., free energy, binding equilibrium and association/dissociation kinetic rate constants) is presented and its applicability to a number of protein-DNA complexes and dsDNA melting demonstrated using authors' own and literature data.
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PMID:Evaluation of mixed-salt effects on thermodynamic and kinetic parameters of RNA polymerase-promoter DNA complexes in terms of equivalent salt concentrations. General applicability to DNA complexes. 1989 92

Reverse transcriptase (RT) catalyzes the formation of dsDNA from single-stranded retroviral RNA genome. This enzyme is unique among DNA polymerases in its ability to use either RNA or DNA as a template. Moloney Murine Leukemia virus reverse transcriptase lacking RNase H activity (M-MLVH- RT) especially holds particular interest because of its ability to eliminate the deleterious effect of RNase H, which results in more efficient synthesis of full-length cDNA from mRNA. Therefore, the development of a simple purification method attracts the attention of retroviral drug and enzyme researchers and manufacturers. The present work is the first purification example of a non-tagged (native) RT by affinity chromatography using synthetic affinity ligands. In this study, the ligand was selected from a structure-biased combinatorial library of dNTP-mimetic ligands, and it was evaluated for its ability to bind and purify M-MLVH- RT from inclusion bodies of recombinant E. coli. The selected ligand (AEAd), bearing 9-aminoethyladenine and 1,6-diamine-hexane both linked on the same triazine scaffold, displayed the highest enzyme purifying ability after applying mild desorption conditions (6 mM MnCl(2) in 20 mM Tris-HCl buffer, pH 7.5). The binding capacity of immobilized AEAd with M-MLVH- RT was determined to be equal to approximately 1 mg enzyme/g moist weight gel. Adsorption studies with immobilized AEAd and soluble M-MLVH- RT demonstrated that the formation of the respective complex was perturbed by ATP. Quality control tests of the purified M-MLVH- RT essentially showed a single band (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and absence of nucleic acids and contaminating nuclease activities.
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PMID:Purification of M-MLVH- RT on a 9-aminoethyladenine-(1,6-diamine-hexane)-triazine selected from a combinatorial library of dNTP-mimetic ligands. 2082 67

Enteric bacteria encounter a wide range of pHs throughout the human intestinal tract. We conducted experimental evolution of Escherichia coli K-12 to isolate clones with increased fitness during growth under acidic conditions (pH 4.5 to 4.8). Twenty-four independent populations of E. coli K-12 W3110 were evolved in LBK medium (10 g/liter tryptone, 5 g/liter yeast extract, 7.45 g/liter KCl) buffered with homopiperazine-N,N'-bis-2-(ethanosulfonic acid) and malate at pH 4.8. At generation 730, the pH was decreased to 4.6 with HCl. By 2,000 generations, all populations had achieved higher endpoint growth than the ancestor at pH 4.6 but not at pH 7.0. All evolving populations showed a progressive loss of activity of lysine decarboxylase (CadA), a major acid stress enzyme. This finding suggests a surprising association between acid adaptation and moderation of an acid stress response. At generation 2,000, eight clones were isolated from four populations, and their genomes were sequenced. Each clone showed between three and eight missense mutations, including one in a subunit of the RNA polymerase holoenzyme (rpoB, rpoC, or rpoD). Missense mutations were found in adiY, the activator of the acid-inducible arginine decarboxylase (adiA), and in gcvP (glycine decarboxylase), a possible acid stress component. For tests of fitness relative to that of the ancestor, lacZ::kan was transduced into each strain. All acid-evolved clones showed a high fitness advantage at pH 4.6. With the cytoplasmic pH depressed by benzoate (at external pH 6.5), acid-evolved clones showed decreased fitness; thus, there was no adaptation to cytoplasmic pH depression. At pH 9.0, acid-evolved clones showed no fitness advantage. Thus, our acid-evolved clones showed a fitness increase specific to low external pH.
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PMID:Acid-adapted strains of Escherichia coli K-12 obtained by experimental evolution. 2555 91

Three species of Sarcocystis-S. miescheriana, S. suihominis, and S. porcifelis-have been recorded from pigs ( Sus scrofa ). Among these 3 species, the zoonotic species S. suihominis is of paramount importance and an important food safety issue. Previous studies indicate prevalence of porcine Sarcocystis species in India, but molecular evidence, among other evidence, is required for proper species differentiation. Myocardium from 250 stray and farm pigs destined for slaughter for human consumption were collected from slaughter shops located in urban slums in Punjab, northern India. Tissues were examined for Sarcocystis by using an intact cyst isolation method, pepsin acid digestion, Sarcocystis 18S ribosomal RNA polymerase chain reaction (PCR), and real-time quantitative PCR with melting curve analysis (qPCR-MCA). The combination of primers was used for 18S rRNA PCR amplification followed by sequencing. Ten representative samples were sequenced in both the directions from which 7 readable sequences were obtained for phylogenetic analysis. Sarcocystis cysts/zoites were recorded in 146 (58.4%), 169 (67.6%), 182 (72.8%), and 191 (76.4%) of samples by using intact cyst isolation, pepsin HCl digestion, conventional PCR, and qPCR-MCA, respectively. Molecularly, 1 S. miescheriana isolate and 6 isolates of the zoonotic species S. suihominis were recorded. This is the first study providing molecular identification for the presence of zoonotic species S. suihomonis in India. The prevalence of zoonotic S. suihominis in pork in India is worrisome and warrants intervention policies to stop the practice of rearing pigs under unhygienic conditions.
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PMID:Pervasive Environmental Contamination with Human Feces Results in High Prevalence of Zoonotic Sarcocystis Infection in Pigs in the Punjab, India. 2658 86


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