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)

"Positive control" mutants of the cI protein of bacteriophage lambda (lambda cI) bind DNA but, unlike the wild-type protein, fail to activate transcription. According to the original interpretation of Ptashne and co-workers, these mutants bear amino acid substitutions that disrupt a stimulatory interaction between lambda cI bound at operator site O(R)2 and RNA polymerase bound at promoter P(RM), an idea supported by kinetic analysis in one case. Genetic analysis has suggested that one residue in particular, glutamate 34 (E34), is critical for the stimulatory effect of wild-type lambda cI. More recently, however, Kolkhof and Muller-Hill have challenged this view, suggesting that mutant E34K fails to activate because it binds at unusually low concentrations to O(R)3, a site that mediates repression of P(RM). To test this hypothesis, we have examined the behaviour of the lambda cI-E34K mutant both in vitro and in vivo by assaying transcription from P(RM) and monitoring operator site occupancy over a range of protein concentrations. Our results are inconsistent with the interpretation of Kolkhof and Muller-Hill, and demonstrate that under conditions where lambda operator O(R)2 is fully occupied and operator O(R)3 is vacant, wild-type lambda cI activates transcription from promoter P(RM) whereas the mutant does not.
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PMID:The activation defect of a lambda cI positive control mutant. 901 40

Transcription of the proP gene, encoding a transporter of the osmoprotectants proline and glycine betaine, is controlled from two promoters, P1 and P2, that respond primarily to osmotic and stationary-phase signals, respectively. The P1 promoter is normally expressed at a very low level under low or normal medium osmolarity. We demonstrate that the binding of the cyclic AMP (cAMP) receptor protein (CRP) to a site centered at -34.5 within the promoter is responsible for the low promoter activity under these conditions. A brief period of reduced CRP binding in early log phase corresponds to a transient burst of P1 transcription upon resumption of growth in Luria-Bertani broth. A CRP binding-site mutation or the absence of a functional crp gene leads to high constitutive expression of P1. We show that the binding of CRP-cAMP inhibits transcription by purified RNA polymerase in vitro at P1, but this repression is relieved at moderately high potassium glutamate concentrations. Likewise, open-complex formation at P1 in vivo is inhibited by the presence of CRP under low-osmolarity conditions. Because P1 expression can be further induced by osmotic upshifts in a delta crp strain or in the presence of the CRP binding-site mutation, additional controls exist to osmotically regulate P1 expression.
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PMID:Cyclic AMP receptor protein functions as a repressor of the osmotically inducible promoter proP P1 in Escherichia coli. 907 29

The proU operon in enterobacteria encodes a binding-protein-dependent transporter for the active uptake of glycine betaine and L-proline, and serves an adaptive role during growth of cells in hyperosmolar environments. Transcription of proU is induced 400-fold under these conditions, but the underlying signal transduction mechanisms are incompletely understood. Increased DNA supercoiling and activation by potassium glutamate have each been proposed in alternative models as mediators of proU osmoresponsivity. We review here the available experimental data on proU regulation, and in particular the roles for DNA supercoiling, potassium glutamate, histone-like proteins of the bacterial nucleoid, and alternative sigma factors of RNA polymerase in such regulation. We also propose a new unifying model, in which the pronounced osmotic regulation of proU expression is achieved through the additive effects of at least three separate mechanisms, each comprised of a cis element [two promoters P1 and P2, and negative-regulatory-element (NRE) downstream of both promoters] and distinct trans-acting factors that interact with it: stationary-phase sigma factor RpoS with P1, nucleoid proteins HU and IHF with P2, and nucleoid protein H-NS with the NRE. In this model, potassium glutamate may activate proU expression through each of the three mechanisms whereas DNA supercoiling has a very limited role, if any, in the osmotic induction of proU transcription. We also suggest that proU may be a virulence gene in the pathogenic enterobacteria.
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PMID:How is osmotic regulation of transcription of the Escherichia coli proU operon achieved? A review and a model. 908 63

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.
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PMID:Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases. 912 28

By combining biochemical, molecular and immunohistochemical approaches, we have investigated the presence of metabotropic glutamate receptors (mGluRs) belonging to the subtype 5 in rat and human spinal cords and the developmental changes in their expression. A polyclonal antibody raised against the carboxy-terminal portion of mGluR5 was used to study the distribution of the receptor in rat foetal (Et15), neonatal (P8) and adult spinal cords and dorsal root ganglia (DRG). mGluR5 appeared to be predominantly expressed in regions containing the primary sensory afferents. Immunoblotting with anti-mGluR5 antibody revealed lower receptor protein levels in rat adult spinal cord when compared with P8 rat spinal cord. Reverse transcriptase-polymerase chain reaction showed both mGluR5a and mGluR5b mRNAs expression in rat spinal cord. The mGluR5a variant was found more abundant in young animals than in adults. The pattern of mGluR5 immunostaining was also studied in foetal (6-8, 10, 12 and 22 weeks of gestation) and adult human spinal cord. At all stages of human development, a strong mGluR5 immunoreactivity was observed in the dorsal roots and in the dorsal and dorsolateral funiculi with maximum levels of staining at week 12 of gestation. Foetal DRG neurons were heterogeneously labeled. mGluR5 was also diffusely detectable in the mantle layer. In adult human spinal cords, immunoreactivity was confined to laminae I and II of the dorsal horns. These results demonstrate for the first time the presence of mGluR5 in human spinal cord. The distribution of this receptor suggests a role in the development of somatosensory pathways and in the control of nociceptive neurotransmission.
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PMID:mGluR5 metabotropic glutamate receptor distribution in rat and human spinal cord: a developmental study. 917 80

The rpoF gene of Escherichia coli codes for the RNA polymerase sigmaF (or sigma28) subunit, which is involved in transcription of the flagellar and chemotaxis genes. Both sigmaF and sigma70 (the major sigma subunit in growing cells) were overexpressed, purified to homogeneity, and compared with respect to activity and specificity. The affinity of sigmaF to core RNA polymerase (E) is higher than that of sigma70, as measured by gel filtration high-pressure liquid chromatography. In an in vitro transcription system, the holoenzyme (E sigmaF) containing sigmaF selectively transcribed the flagellar and chemotaxis genes, all of which could not be transcribed by E sigma70. This strict promoter recognition property of sigmaF is similar to those of other stress response minor sigma subunits but different from those of the principal sigma subunits, sigma70 and sigma38. sigma70-dependent transcription in vitro is inhibited at high concentrations of all salts tested, showing maximum activity at 50 mM. In contrast, sigmaF-dependent transcription was maximum at 50 mM KCI and then decreased to negligible level at 300 mM; in the cases of potassium acetate and potassium glutamate, maximum transcription was between 200 and 300 mM. DNase I foot printing of the fliC and fliD promoters indicated that sigmaF alone is unable to bind DNA, but E sigmaF specifically recognizes -10 and -35 regions of the sigmaF-dependent promoters with rather long upstream protection. Alteration of the promoter structure after binding of E sigmaF was suggested.
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PMID:Promoter selectivity of Escherichia coli RNA polymerase sigmaF holoenzyme involved in transcription of flagellar and chemotaxis genes. 920 42

The effect of potassium glutamate was examined on the DNA-directed in vitro protein synthesizing system of Salmonella typhimurium which conventionally contained acetate as a sole counter anion. The glutamate replacement increased the potassium optimum by about 70% and improved the expression of different DNA templates, but selectively. The biggest improvements in expression (about 8-fold) were seen with a lacUV5 (from Escherichia coli) template and with a mutant promoter his operon (from S. typhimurium) template. In contrast, the expression of a leuV promoter (from Escherichia coli) template was relatively unaffected by the glutamate replacement. The chain-growth-rate of mRNA and polypeptide syntheses in the DNA-directed in vitro protein synthesizing system were unaffected by the glutamate replacement. It was concluded that at least a part of the effect of glutamate replacement is on RNA polymerase-promoter interaction, and most likely the association step. Glutamate replacement did not alter the ppGpp-mediated positive and negative regulation of the his and leuV promoter, respectively, in the in vitro system. Taken together, the results suggest that the use of potassium glutamate in place of potassium acetate in DNA-directed in vitro synthesis provides a physiologically more relevant approximation of the ionic environment in vivo.
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PMID:The DNA-directed in vitro protein synthesizing system of Salmonella typhimurium: the effect of glutamate substitution. 925 65

Here we report the cloning and functional analysis of a cDNA encoding a functional glutamate receptor subunit of Oreochromis sp., a freshwater teleost fish. The deduced amino acid sequence of this cDNA clone, fGluR3 alpha, displays the highest sequence identity to that of the mammalian GluR3 subunit. Results of quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis indicated that the expression level of fGluR3 alpha in the cerebellum was much less than that in the telencephalon and optical lobe. Similar to its mammalian counterpart, variants of fGluR3 alpha were created by alternative splicing and RNA editing at the R/G site. The channel properties of homomeric fGluR3 alpha expressed in Xenopus oocytes were similar to those of the mammalian alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-preferring receptors. The rank order of agonist potency of the expressed fGluR3 alpha is AMPA > or = glutamate > or = quisqualate > domoate > or = kainate. This is the first functional glutamate receptor of teleost fish being demonstrated to be sensitive to AMPA. Furthermore, this study suggested a strong functional conservation of AMPA-preferring receptors in vertebrates.
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PMID:Molecular and electrophysiological characterizations of fGluR3 alpha, an ionotropic glutamate receptor subunit of a teleost fish. 967 19

Cultured astrocytes derived from neonatal rat brain exhibited high affinity, Na+-dependent, paroxetine and fluoxetine sensitive [3H]5-HT uptake. Reverse transcriptase-PCR demonstrated that astrocytes in culture expressed messenger RNA for the cloned serotonin transporter protein which has been characterised as the neuronal serotonin transporter. Although the serotonin transporter in cultured astrocytes displayed a Km value approximately 10 times greater than found in adult brain synaptosomes, these observations indicated that astrocytes in vitro may express the same serotonin transporter as neurons. Reverse transcriptase-PCR demonstrated the presence of serotonin transporter mRNA in the adult rat cerebral cortex, suggesting that astrocytes in vivo may express low levels of this mRNA. To investigate whether astrocytes in the adult CNS express functional serotonin transporters, glial plasmalemmal vesicles were prepared from cerebral cortex, representing a subcellular fraction composed primarily of vesicles derived from astrocytes. These vesicles were characterised by [3H]-glutamate and [3H]-dopamine uptake and by immunoblot analysis, using glial and synaptic markers: glutamate synthase, SNAP-25 and synaptobrevin. [3H]5-HT was taken up into glial plasmalemmal vesicles in a high affinity (Km approximately 40 nM), Na+ dependent, paroxetine-sensitive manner. The [3H]5-HT uptake capacity (Vmax) in these vesicles was approximately one quarter of that observed in synaptosomes. These data indicate that astrocytes in culture and in vivo are capable of 5-HT uptake via the previously characterised 'neuronal' serotonin transporter.
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PMID:Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles. 969 37

The most potent promoters in the ddlB-ftsA region of the dcw cluster have been analysed for sigmaS-dependent transcription. Only the gearbox promoter ftsQ1p was found to be transcribed in vitro by RNA polymerase holoenzyme coupled to sigmaS (EsigmaS). This dependency on sigmaS was also found in vivo when single-copy fusions to a reporter gene were analysed in rpoS and rpoS+ backgrounds. Although ftsQ1p can be transcribed by RNA polymerase containing either sigmaD or sigmaS, there is a preference for EsigmaS when the assay conditions include potassium glutamate and supercoiled templates, a property shared with the bolA1p gearbox promoter. The rest of the promoters assayed, ftsQ2p and ftsZ2p3p4p, similarly to the control bolA2p promoter, were preferentially transcribed by EsigmaD, the housekeeper polymerase. The ftsQ1p and the bolA1p promoters also share the presence of AT-rich sequences upstream of the - 35 region and the requirement for an intact wild-type alpha-subunit for a proficient transcription, allowing their joint classification as gearboxes.
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PMID:The ftsQ1p gearbox promoter of Escherichia coli is a major sigma S-dependent promoter in the ddlB-ftsA region. 979 Nov 85


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