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)

Antibiotics are very commonly used substances to eradicate bacterial infections by bacteriostatic or even bactericid effect. They act at a very specific stage (target), although other less important or secondary interactions can occur. We studied the interaction of three antibiotic families (beta-lactamins, aminosides, rifampicin) with bacterial cell. Penicillin disturbs the cell wall synthesis and more accurately the glycopeptide (or murein) formation, a substance giving rigidity or shape to bacteria. It acts in the late phase of murein-biosynthesis, when N-acetyl glucosamin -- N-acetyl muramic acid L ala -D glu M-DAP (L lys) -D ala -D ala are linked together by the peptide part, under the effect of several enzymes, particularly transpeptidase and DD-carboxy-peptidase. It would appear that beta-lactame-thiazolidine rings have a steric analogy with dipeptide D-alanyl D-alanine. The result would be that the enzyme would act on the antibiotic instead of peptide: the consequence would be inhibition of the peptidic link, giving an abnormal murein, and an incomplete cell wall i.e. fragile bacteria. Aminosides, particularly Streptomycin, link themselves to 30 S subunit of bacterial ribosome. In this case, it seems that it is a 3''OH function which reacts with lysine (from S 12 protein part of 30 S subunit). The consequence is an alteration in the RNA messager lecture, and a false traduction and consequently protein biosynthesis stops with a decrease of polyribosomes and of the formation of inert 70 S ribosome. Rifamycins, and particularly Rifampicin act by inhibition of RNA messager synthesis. One molecule of antibiotic links itself to one molecule of RNA messager : hydroxyl and cetone function in C1 Cs C21 C23 and "ansa" bridge link to beta subunit of RNA polymerase. This linkage gives a conformational change to the RNA polymerase-DNA complex, inhibiting the catalytic action of this enzyme, and consequently stopping RNA messager and protein synthesis. The study of the action mechanism of these antibiotics enables us to show the action specificity of these products in the bacteria. This specificity is more accurate when the target is not to be found in the eucaryotic cells : in this case the antibiotic may be considered as entirely atoxic. If the study of the action mechanism of antibiotics gives a better understanding of the use of these drugs, their action at a definite stage in bacterial metabolism is a valuable tool for scientists in their approach to cell functioning.
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PMID:[Mechanism of action of antibiotics:some examples]. 15 42

Lysine 274 is conserved in all known fructose-1,6-bisphosphatase sequences. It has been implicated in substrate binding and/or catalysis on the basis of reactivity with pyridoxal phosphate as well as by x-ray crystallographic analysis. Lys274 of rat liver fructose-1,6-bisphosphatase was mutated to alanine by the polymerase chain reaction, and the T7-RNA polymerase-transcribed construct containing the mutant sequence was expressed in Escherichia coli. The mutant and wild-type forms of the enzyme were purified to homogeneity, and their specific activity, substrate dependence, and inhibition by fructose 2,6-bisphosphate and AMP were compared. While the mutant exhibited no change in maximal velocity, its Km for fructose 1,6-bisphosphate was 20-fold higher than that of the wild-type, and its Ki for fructose 2,6-bisphosphate was increased 1000-fold. Consistent with the unaltered maximal velocity, there were no apparent difference between the secondary structure of the wild-type and mutant enzyme forms, as measured by circular dichroism and ultraviolet difference spectroscopy. The Ki for the allosteric inhibitor AMP was only slightly increased, indicating that Lys274 is not directly involved in AMP inhibition. Fructose 2,6-bisphosphate potentiated AMP inhibition of both forms, but 500-fold higher concentrations of fructose 2,6-bisphosphate were needed to reduce the Ki for AMP for the mutant compared to the wild-type. However, potentiation of AMP inhibition of the Lys274----Ala mutant was evident at fructose 2,6-bisphosphate concentrations (approximately 100 microM) well below those that inhibited the enzyme, which suggests that fructose 2,6-bisphosphate interacts either with the AMP site directly or with other residues involved in the active site-AMP synergy. The results also demonstrate that although Lys274 is an important binding site determinant for sugar bisphosphates, it plays a more significant role in binding fructose 2,6-bisphosphate than fructose 1,6-bisphosphate, probably because it binds the 2-phospho group of the former while other residues bind the 1-phospho group of the substrate. It is concluded that the enzyme utilizes Lys274 to discriminate between its substrate and fructose 2,6-bisphosphate.
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PMID:Lysine 274 is essential for fructose 2,6-bisphosphate inhibition of fructose-1,6-bisphosphatase. 131 10

The fructose-2,6-bisphosphatase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been shown to be structurally and functionally homologous to phosphoglycerate mutase. Both enzymes catalyze their reactions via phosphoenzyme intermediates which utilize an active site histidine as a nucleophilic phosphoacceptor and another histidine as a proton donor to the leaving group. Glu327 in the bisphosphatase domain of the rat liver bifunctional enzyme is conserved in all phosphoglycerate mutase structures and is postulated, by modelling studies, to be located in the active site. Glu327 was mutated to Ala, Gln, or Asp. The mutant and wild-type enzymes were expressed in Escherichia coli with a T-7 RNA polymerase-based expression system and purified to homogeneity by substrate elution from phosphocellulose. The Glu327 mutants had apparent molecular weights of 110,000 by gel filtration and had unaltered 6-phosphofructo-2-kinase activity. Circular dichroism showed that the secondary structure of the Glu327 mutant enzyme forms was the same as the wild-type enzyme. The maximal velocity of the fructose-2,6-bisphosphatase of the Glu327----Ala, Glu327----Gln, and Glu327----Asp mutants was 4, 2, and 20%, respectively, that of the wild-type enzyme, but the rate of phosphoenzyme formation of the mutants was reduced by at least a factor of 1000. In addition, the rate constants of phosphoenzyme hydrolysis for the Glu372----Ala and Glu327----Gln mutants were 2.7 and 1.3%, respectively, of the wild type, whereas the rate constant for the Glu327----Asp mutant was 60% of the wild-type value. Glu327 was not a substrate or product binding site determinant since the Km for fructose-2,6-bisphosphate and Ki for fructose-6-phosphate of the mutants were not appreciably changed. The results implicate Glu327 as part of a catalytic triad in fructose-2,6-bisphosphatase and suggest that it influences the protonation state of the active site histidine residues during phosphoenzyme formation and/or acts as a base catalyst to enhance the nucleophilic attack of water on the phosphoenzyme intermediate.
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PMID:Glu327 is part of a catalytic triad in rat liver fructose-2,6-bisphosphatase. 131 12

The high affinity branched-chain amino acid transport system (LIV-I) in Pseudomonas aeruginosa is composed of five components: BraC, a periplasmic binding protein for branched-chain amino acids; BraD and BraE, integral membrane proteins; BraF and BraG, putative nucleotide-binding proteins. By using a T7 RNA polymerase/promoter system we overproduced the BraD, BraE, BraF, and BraG proteins in Escherichia coli. The proteins were found to form a complex in the E. coli membrane and solubilized from the membrane with octyl glucoside. The LIV-I transport system was reconstituted into proteoliposomes from solubilized proteins by a detergent dilution procedure. In this reconstituted system, leucine transport was completely dependent on the presence of all five Bra components and on ATP loaded internally to the proteoliposomes. Alanine and threonine in addition to branched-chain amino acids were transported by the proteoliposomes, reflecting the substrate specificity of the BraC protein. GTP replaced ATP well as an energy source, and CTP and UTP also replaced ATP partially. Consumption of loaded ATP and concomitant production of orthophosphate were observed only when BraC and leucine, a substrate for LIV-I, were added together to the proteoliposomes, indicating that the LIV-I transport system has an ATPase activity coupled to translocation of branched-chain amino acids across the membrane.
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PMID:Solubilization and reconstitution of the Pseudomonas aeruginosa high affinity branched-chain amino acid transport system. 140 Apr 43

The P2 ogr gene encodes a 72-amino-acid protein required for P2 late gene expression. This gene was defined originally by a class of compensatory mutations which overcome the block to P2 late transcription imposed by a host mutation, rpoA109, in the gene encoding the alpha subunit of Escherichia coli RNA polymerase. Spontaneous compensatory ogr mutations substitute a Cys for a Tyr residue at amino acid 42 in the Ogr polypeptide. Using suppression of an ogr amber mutation and site-directed oligonucleotide mutagenesis, we have studied the effect of amino acid substitutions at this position in Ogr. Substitution of charged residues at this site renders Ogr protein inactive, in rpoA+ and rpoA109 strains. While 11 different amino acids are capable of replacing the wild-type Tyr-42 to allow P2 growth to varying degrees in a wild-type E. coli strain, only three of these allow phage growth in strains carrying the rpoA109 mutation. Phages carrying Cys or Ala in place of Tyr-42 gave burst sizes at least as high as P2 ogr+ in a rpoA+ strain; a Gly substitution also allowed P2 to grow in either a rpoA+ or rpoA109 background, but markedly reduced the burst size. These results are consistent with a direct interaction between Ogr and the alpha subunit of E. coli RNA polymerase in positive control of P2 late transcription, and indicate that the block imposed by the rpoA109 mutation is due to steric hindrance.
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PMID:Site-directed mutagenesis of an amino acid residue in the bacteriophage P2 ogr protein implicated in interaction with Escherichia coli RNA polymerase. 148 87

RNA-dependent RNA polymerases contain a highly conserved region of amino acids with a core segment composed of the amino acids YGDD which have been hypothesized to be at or near the catalytic active site of the molecule. Six mutations in this conserved YGDD region of the poliovirus RNA-dependent RNA polymerase were made by using oligonucleotide site-directed DNA mutagenesis of the poliovirus cDNA to substitute A, C, M, P, S, or V for the amino acid G. The mutant polymerase genes were expressed in Escherichia coli, and the purified RNA polymerases were tested for in vitro enzyme activity. Two of the mutant RNA polymerases (those in which the glycine residue was replaced with alanine or serine) exhibited in vitro enzymatic activity ranging from 5 to 20% of wild-type activity, while the remaining mutant RNA polymerases were inactive. Alterations in the in vitro reaction conditions by modification of temperature, metal ion concentration, or pH resulted in no significant differences in the activities of the mutant RNA polymerases relative to that of the wild-type enzyme. An antipeptide antibody directed against the wild-type core amino acid segment containing the YGDD region of the poliovirus polymerase reacted with the wild-type recombinant RNA polymerase and to a limited extent with the two enzymatically active mutant polymerases; the antipeptide antibody did not react with the mutant RNA polymerases which did not have in vitro enzyme activity. These results are discussed in the context of secondary-structure predictions for the core segment containing the conserved YGDD amino acids in the poliovirus RNA polymerase.
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PMID:Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif. 165 2

During sporulation in Bacillus subtilis, new RNA polymerase sigma factors are produced. These sigma factors direct the transcription of genes that are required for this cellular differentiation. In order to determine the role of each sigma factor in this process, it is necessary to know which promoters are recognized by each sigma factor. The spoIIID gene product plays an important role in the establishment of mother cell-specific gene expression during sporulation. We found that substitution of an alanine at position 124 of the sporulation-specific sigma factor sigma E suppressed the effect of a single-base-pair transition at position -13 of the spoIIID promoter. This alanine substitution in sigma E did not suppress the effect of a transversion at position -12 of the spoIIID promoter. The allele specificity of the interaction between sigma E and the spoIIID promoter is strong evidence that sigma E directs transcription from the spoIIID promoter during sporulation. Position 124 in sigma E is located within a region that is highly conserved among the regions in other sigma factors that probably interact with the -10 regions of their cognate promoters.
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PMID:Genetic evidence for interaction of sigma E with the spoIIID promoter in Bacillus subtilis. 174 38

The active center of DNA-dependent RNA polymerase performs the principal biochemical reaction of gene expression. Using cross-linkable substrate analogs and site-directed mutations, two evolutionarily invariant amino acids in the beta subunit of the Escherichia coli enzyme (Lys1065 and His1237) were mapped close to the binding site of the priming substrate of the reaction. Surprisingly, the mutational substitution of these residues (Lys1065----Arg and His1237----Ala) did not inactivate the catalytic function, but inhibited transition from the initiation to the elongation stage of transcription.
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PMID:Mapping of the priming substrate contacts in the active center of Escherichia coli RNA polymerase. 174 65

The nusA11 mutation causes reduced transcription termination and temperature-sensitive growth of Escherichia coli. Suppressor mutations that restored growth of nusA11 mutant cells were isolated and named sna mutations. The intergenic suppressor mutation sna-10 was located in the rpoC gene at 90 min, which encodes the beta' subunit of RNA polymerase. sna-10 complemented the defect in tR1 termination caused by nusA11 and by itself stimulated termination of transcription at the lambda tR1 terminator. sna-10 is specific to the nusA11 allele and unable to suppress cold-sensitive growth of the nusA10 mutant. nusA10 carried two base substitutions at positions 311 and 634, causing two amino acid changes from the wild-type sequence. During these studies, we found three -1 frameshift errors in the wild-type nusA sequence; the correct sequence was confirmed by the peptide sequence and gene fusion analyses. The revised sequence revealed that nusA1 and nusA11 are located in an arginine-rich peptide region and substitute arginine and aspartate for leucine 183 and glycine 181, respectively. The intragenic suppressor study indicated that the nusA11 mutation can be suppressed by changing the mutated aspartate 181 to alanine or changing aspartate 84 to tyrosine.
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PMID:Genetic interaction between the beta' subunit of RNA polymerase and the arginine-rich domain of Escherichia coli nusA protein. 184 65

The importance of certain amino acid residues in mammalian ornithine decarboxylase activity and degradation was studied by site-specific mutagenesis. Changes were made to the mouse ornithine decarboxylase cDNA in a plasmid containing a T7 RNA polymerase promoter. The plasmid was then used for the synthesis of RNA, which was translated in a reticulocyte lysate system. The activity of the ornithine decarboxylase formed and the stability of the protein to degradation in a reticulocyte lysate system were determined. Changes of lysine-169 or of histidine-197 to alanine completely abolished enzyme activity, indicating that these residues are essential for enzyme activity. The removal of the C-terminal 36 residues, the mutation of lysine-349 to alanine, of lysine-298 to alanine or the double change of serine-303 and glutamic acid-308 to alanine residues still resulted in an active enzyme. The last-mentioned finding indicates that the phosphorylation of serine-303 does not play an essential role in the catalytic activity of ornithine decarboxylase. The control ornithine decarboxylase protein was degraded rapidly in a reticulocyte lysate provided that ATP was added. The truncated protein missing the 36 residues from the C-terminus was much more stable in this system, and the protein containing the double change of serine-303 and glutamic acid-308 to alanine residues was slightly more stable than control ornithine decarboxylase protein. These results indicate that the altered residues may play a role in interaction with factors responsible for the rapid turnover of ornithine decarboxylase.
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PMID:Identification of residues in ornithine decarboxylase essential for enzymic activity and for rapid protein turnover. 187 2


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