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)

Synthetic analogs of the 5'-terminal caps of eukaryotic mRNAs and snRNAs are used in elucidating such physiological processes as mRNA translation, pre-mRNA splicing, intracellular transport of mRNA and snRNAs, and mRNA turnover. Particularly useful are RNAs capped with synthetic analogs, which are produced by in vitro transcription of a DNA template using a bacteriophage RNA polymerase in the presence of ribonucleoside triphosphates and a cap dinucleotide such as m(7)Gp(3)G. Unfortunately, because of the presence of a 3'-OH on both the m(7)Guo and Guo moieties, up to half of the mRNAs contain caps incorporated in the reverse orientation. Previously we designed and synthesized two "anti-reverse" cap analogs (ARCAs), m(7)3'dGp(3)G and m(2)(7,3'-)(O)Gp(3)G, that cannot be incorporated in the reverse orientation because of modifications at the C3' position of m(7)Guo. In the present study, we have synthesized seven new cap analogs modified in the C2' and C3' positions of m(7)Guo and in the number of phosphate residues, m(2)(7,2'-)(O)Gp(3)G, m(7)2'dGp(3)G, m(7)2'dGp(4)G, m(2)(7,2'-)(O)Gp(4)G, m(2)(7,3'-)(O)Gp(4)G, m(7)Gp(5)G, and m(2)(7,3'-)(O)Gp(5)G. These were analyzed for conformation in solution, binding affinity to eIF4E, inhibition of in vitro translation, degree of reverse capping during in vitro transcription, capping efficiency, and the ability to stimulate cap-dependent translation in vitro when incorporated into mRNA. The results indicate that modifications at C2', like those at C3', prevent reverse incorporation, that tetra- and pentaphosphate cap analogs bind eIF4E and inhibit translation more strongly than their triphosphate counterparts, and that tetraphosphate ARCAs promote cap-dependent translation more effectively than previous cap analogs.
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PMID:Novel "anti-reverse" cap analogs with superior translational properties. 1292 59

The nitrogen-fixing symbiosis between Sinorhizobium meliloti and Medicago sativa requires complex physiological adaptation by both partners. One method by which bacteria coordinately control physiological adaptation is the stringent response, which is triggered by the presence of the nucleotide guanosine tetraphosphate (ppGpp). ppGpp, produced by the RelA enzyme, is thought to bind to and alter the ability of RNA polymerase (RNAP) to initiate and elongate transcription and affect the affinity of the core enzyme for various sigma factors. An S. meliloti relA mutant which cannot produce ppGpp was previously shown to be defective in the ability to form nodules. This mutant also overproduces a symbiotically necessary exopolysaccharide called succinoglycan. The work presented here encompasses the analysis of suppressor mutants, isolated from host plants, that suppress the symbiotic defects of the relA mutant. All suppressor mutations are extragenic and map to either rpoB or rpoC, which encode the beta and beta' subunits of RNAP. Phenotypic, structural, and gene expression analyses reveal that suppressor mutants can be divided into two classes; one is specific in its effect on stringent response-regulated genes and shares striking similarity with suppressor mutants of Escherichia coli strains that lack ppGpp, and another reduces transcription of all genes tested in comparison to that in the relA parent strain. Our findings indicate that the ability to successfully establish symbiosis is tightly coupled with the bacteria's ability to undergo global physiological adjustment via the stringent response.
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PMID:Mutations in rpoBC suppress the defects of a Sinorhizobium meliloti relA mutant. 1294 13

Guanosine-tetraphosphate (ppGpp) is a major regulator of stringent control, an adaptive response of bacteria to amino acid starvation. The 2.7 A resolution structure of the Thermus thermophilus RNA polymerase (RNAP) holoenzyme in complex with ppGpp reveals that ppGpp binds to the same site near the active center in both independent RNAP molecules in the crystal but in strikingly distinct orientations. Binding is symmetrical with respect to the two diphosphates of ppGpp and is relaxed with respect to the orientation of the nucleotide base. Different modes of ppGpp binding are coupled with asymmetry of the active site configurations. The results suggest that base pairing of ppGpp with cytosines in the nontemplate DNA strand might be an essential component of transcription control by ppGpp. We present experimental evidence highlighting the importance of base-specific contacts between ppGpp and specific cytosine residues during both transcription initiation and elongation.
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PMID:Structural basis for transcription regulation by alarmone ppGpp. 1510 91

Guanosine tetraphosphate (ppGpp) is a signal of nutritional stress that regulates transcription. An RNA polymerase rudder mutant rpoC (Delta 312-315) is found to suppress ppGpp deficiency phenotypes by restoring both negative and positive activities of promoter fusions in vivo, as if ppGpp were present. Measurements of defects in transcription of the PargT tRNA promoter with mutant RNA polymerase reveal that the mutant enzyme quantitatively mimics the presence of added ppGpp. DNaseI footprints and mobility shifts under RNA polymerization conditions reveal that the promoter-specific transcription defect of the mutant enzyme can be ascribed to the presence of inactive dead-end promoter complexes with features similar to those of a stable closed complex. We propose that formation of such inactive complexes represents an alternative explanation of "stringent RNA polymerase" mutant behavior to those currently published, and it represents a newly discovered mode of action of ppGpp.
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PMID:Conversion of active promoter-RNA polymerase complexes into inactive promoter bound complexes in E. coli by the transcription effector, ppGpp. 1578 Sep 38

A new method for determination of RNA polymerase (RNAP) activity is presented. The method uses nucleoside tri- and tetraphosphate derivatives carrying 4-methylumbelliferone residue at the terminal phosphate. Incorporation of such compounds in RNA by RNA polymerase is accompanied by release of di- and triphosphate derivatives of 4-methylumbelliferone. Subsequent treatment by alkaline phosphatase produces free 4-methylumbelliferone that is highly fluorescent and can be easily detected. The sensitivity of the method is higher than that reported in previous studies. The validity of the assay has been demonstrated by retrieving the RNAP inhibitors from a collection of 16,000 compounds.
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PMID:Homogeneous fluorescent assay for RNA polymerase. 1595 Jan 66

The RNA polymerase-binding protein DksA is a cofactor required for guanosine tetraphosphate (ppGpp)-responsive control of transcription from sigma70 promoters. Here we present evidence: (i) that both DksA and ppGpp are required for in vivo sigma54 transcription even though they do not have any major direct effects on sigma54 transcription in reconstituted in vitro transcription and sigma-factor competition assays, (ii) that previously defined mutations rendering the housekeeping sigma70 less effective at competing with sigma54 for limiting amounts of core RNA polymerase similarly suppress the requirement for DksA and ppGpp in vivo and (iii) that the extent to which ppGpp and DksA affect transcription from sigma54 promoters in vivo reflects the innate affinity of the promoters for sigma54-RNA polymerase holoenzyme in vitro. Based on these findings, we propose a passive model for ppGpp/DksA regulation of sigma54-dependent transcription that depends on the potent negative effects of these regulatory molecules on transcription from powerful stringently regulated sigma70 promoters.
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PMID:The guanosine tetraphosphate (ppGpp) alarmone, DksA and promoter affinity for RNA polymerase in regulation of sigma-dependent transcription. 1662 75

Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA(Ser) aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).
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PMID:Physiological analysis of the stringent response elicited in an extreme thermophilic bacterium, Thermus thermophilus. 1701 50

A major challenge in systems biology is to integrate our mechanistic understanding of gene regulation to predict quantitatively how cells will respond to environmental changes. Living cells respond rapidly to the availability of nutrients in part by altering production of ribosomal RNA (rRNA), a limiting component in the biosynthesis of ribosomes. Studies of rRNA transcription by the RNA polymerase of Escherichia coli have identified regulatory roles for guanosine tetraphosphate (ppGpp), the initiating nucleotide, and the protein DksA. To what extent findings from in vitro studies can be used to quantitatively predict in vivo responses to changing nutrient environments is unknown. We developed a mechanistic mathematical model for rRNA transcriptional responses to such changes. Our model accounts for binding of RNAP to its rRNA promoter to form a closed complex, isomerization from a closed complex to an open complex, reversible incorporation of the initiating NTP (iNTP), transcript elongation, and clearance of the promoter. Further, the model incorporates interactions between ppGpp and DksA with transcription intermediates, and it includes an empirical correction to account for salt effects. The model biophysical parameters were determined using 33 single- and multi-round transcription experiments spanning 487 in vitro measurements. By incorporating in vivo measurements of ppGpp and ATP, the model correctly predicted rRNA production rates for cellular responses to nutrient upshifts, downshifts, and outgrowth into fresh medium. Inclusion of DksA was essential in all three cases. Our work provides a foundation for using data-driven computational models to predict the kinetics of in vivo transcriptional responses.
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PMID:Rapid responses of ribosomal RNA synthesis to nutrient shifts. 1721 53

Bacillus subtilis and Streptomyces spp. provide tractable experimental systems for studying cellular responses to adverse environmental conditions. During conditions of extreme nutrient limitation, these prokaryotes exhibit a wide range of adaptations, including the production and secretion of antibiotics and enzymes and the formation of aerial mycelium and spores. In response to these conditions, all bacteria, but not eukaryotic microorganisms, exhibit a "stringent response," during which the unusual guanosine tetraphosphate, ppGpp, accumulates intracellularly. This is accompanied by a marked reduction in the GTP pool, due to ppGpp inhibition of IMP-dehydrogenase, and immediate repression of rRNA synthesis, due to the binding of ppGpp to RNA polymerase. This review summarizes our studies on the bacterial stringent response and its use in applied microbiology. We found that morphological differentiation results from a decrease in the pool of GTP, whereas physiological differentiation (antibiotic production) results from a more direct function of ppGpp. That is, we found that the Streptomyces GTP-binding protein Obg functions by sensing intracellular GTP levels and that certain mutations in the RNA polymerase beta-subunit circumvent dependence on ppGpp in antibiotic production. X-ray crystallographic analysis provided a structural basis for the ppGpp regulation of transcription. On the basis of these findings, we have developed the novel concept of "ribosome engineering," focusing on activation of dormant genes to elicit cellular function fully. Ribosome engineering can be applied to strain improvement, screening of novel metabolites, plant breeding, cell-free translation systems, and the treatment of tuberculosis.
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PMID:From microbial differentiation to ribosome engineering. 1758 68

Although in bacterial cells all genes are transcribed by RNA polymerase, there are 2 additional enzymes capable of catalyzing RNA synthesis: poly(A) polymerase I, which adds poly(A) residues to transcripts, and primase, which produces primers for DNA replication. Mechanisms of actions of these 3 RNA-synthesizing enzymes were investigated for many years, and schemes of their regulations have been proposed and generally accepted. Nevertheless, recent discoveries indicated that apart from well-understood mechanisms, there are additional regulatory processes, beyond the established schemes, which allow bacterial cells to respond to changing environmental and physiological conditions. These newly discovered mechanisms, which are discussed in this review, include: (i) specific regulation of gene expression by RNA polyadenylation, (ii) control of DNA replication by interactions of the starvation alarmones, guanosine pentaphosphate and guanosine tetraphosphate, (p)ppGpp, with DnaG primase, (iii) a role for the DksA protein in ppGpp-mediated regulation of transcription, (iv) allosteric modulation of the RNA polymerase catalytic reaction by specific inhibitors of transcription, rifamycins, (v) stimulation of transcription initiation by proteins binding downstream of the promoter sequences, and (vi) promoter-dependent control of transcription antitermination efficiency.
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PMID:Mechanisms of physiological regulation of RNA synthesis in bacteria: new discoveries breaking old schemes. 1766 83


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