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)

The NTRC protein of enteric bacteria is an enhancer-binding protein that activates transcription by the sigma 54-holoenzyme form of RNA polymerase under nitrogen-limiting conditions. In vitro NTRC must be phosphorylated to catalyze ATP hydrolysis and activate transcription. The site of phosphorylation of NTRC from Salmonella typhimurium is Aspartate 54, which lies in the amino-terminal regulatory domain of the protein. We used site-directed mutagenesis to make "conservative" substitutions at residue 54 to alanine, asparagine, and glutamate, and examined the properties of the mutant NTRC proteins in vitro and in vivo. In vitro none of them was detectably phosphorylated, as expected if D54 is, in fact, the sole site of phosphorylation. D54A and D54N did not activate transcription of glnA but, interestingly, D54E activated constitutively. Activation by D54E was partial compared to that by phosphorylated wild-type NTRC. Combining D54A or D54N with S160F, a change in the central domain of NTRC that partially bypasses the requirement for phosphorylation, yielded doubly mutant proteins that were as active as a form carrying S160F alone, indicating that the changes in D54 did not adversely affect the function of the remainder of NTRC. Combining D54E with S160F increased the levels of constitutive ATPase activity and transcriptional activation above those of mutant NTRC proteins carrying either single change alone. We conclude that phosphorylation of aspartate 54 is required to activate NTRC and postulate that the D54E mutation mimics phosphorylation, thereby allowing NTRC to hydrolyze ATP and activate transcription. Phenotypes of mutant strains encoding NTRC proteins with substitutions at D54 indicated that phosphorylation of NTRC at position 54 was necessary for normal growth in the absence of glutamine and that such phosphorylation occurred to some extent even in the absence of NTRB.
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PMID:Glutamate at the site of phosphorylation of nitrogen-regulatory protein NTRC mimics aspartyl-phosphate and activates the protein. 833 71

The DNA binding affinities of several gene-regulatory proteins, restriction endonucleases and the Escherichia coli RNA polymerase have previously been found to be dependent on the nature of the dominant buffer anion. To discover whether the E. coli cAMP receptor protein (CAP) exhibits a similar dependency, we measured its affinity for its primary lactose promoter binding site (lac site 1) in buffers in which the principal anion was chloride, phosphate, sulfate, acetate, or glutamate. We found that the affinity of CAP for lac site 1 is affected only slightly by changes in the dominant buffer anion. The binding of cAMP is similarly insensitive to buffer anion type, indicating that specific protein-anion interactions, if they occur, must be similar for the free and cAMP-bound forms of the protein. The effect of anion substitution on the ability of acrylamide to quench the intrinsic fluorescence of tryptophanyl residues of CAP is also small, suggesting that changes in buffer anion composition have minimal effect on the conformation of tryptophan-proximal regions of CAP. This conclusion is extended by the finding that anion substitution has a relatively small effect on the urea-concentration dependence of CAP denaturation. Taken together, these results support the notion that neither CAP nor CAP.cAMP nor the CAP.cAMP complex with lac promoter DNA interact selectively with anions present in the surrounding buffer. A possible role for this anion-insensitivity in the in vivo function of CAP is suggested.
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PMID:Effects of anions on the binding of the cAMP receptor protein to the lactose promoter. 838 49

The upstream binding factor, UBF, is an RNA polymerase I transcription factor which contains multiple DNA binding domains and a novel protein dimerization domain. Active UBF forms homodimers in vivo through the intramolecular interactions of its dimerization domain, which spans a hundred amino-terminal residues. In the presence of both UBF dimerization domain and its immediately adjacent lysine-rich basic DNA binding domain, the E. coli expressed recombinant polypeptide, dbUBF (dimerization plus basic motifs of UBF), forms homodimers in vitro and binds to double-stranded DNA nonselectively. In gel retardation assay, dbUBF dimers make multiple shift-ladders corresponding to numerous protein dimer-DNA complexes. The UBF dimerization domain contains multiple helical structures, as predicted by EMBO-PHD program. Most of hydrophobic residues in the dimerization domain are confined in the hydrophobic phase of these hypothetic helices. Mutating these hydrophobic residues to glutamate prohibits dbUBF association and gives a different shift pattern in gel retardation assay. The results we present here argue that UBF association is largely exerted by the hydrophobic interactions between the multiple helices to bring two molecules together.
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PMID:The dimerization domain upstream binding factor contains multiple helical structures. 860 48

In this study, the relative abundance of splicing variants of Oreochromis non-NMDA subtype glutamate receptors was studied by quantitative reverse-transcriptase PCR (RT-PCR). The relative expression level between the flip and flop transcripts of fGluR2 alpha determined by quantitative RT-PCR is apparently much higher than that estimated by sequence analysis of the cloned RT-PCR products. Control studies were performed to demonstrate the accuracy of the application of quantitative RT-PCR analysis in studying the relative abundance between the flip and flop transcripts of glutamate receptors.
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PMID:Determination of relative abundance of splicing variants of Oreochromis glutamate receptors by quantitative reverse-transcriptase PCR. 870 49

The largest subunit of RNA polymerase II contains a repetitive C-terminal domain (CTD) consisting of tandem repeats of the consenus sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. Substitution of nonphosphorylatable amino acids at positions two or five of the Saccharomyces cerevisiae CTD is lethal. We developed a selection system for isolating suppressors of this lethal phenotype and cloned a gene, SCA1 (suppressor of CTD alanine), which complements recessive suppressors of lethal multiple-substitution mutations. A partial deletion of SCA1 (sca1 delta ::hisG) suppresses alanine or glutamate substitutions at position two of the consensus CTD sequence, and a lethal CTD truncation mutation, but SCA1 deletion does not suppress alanine or glutamate substitutions at position five. SCA1 is identical to SRB9, a suppressor of a cold-sensitive CTD truncation mutation. Strains carrying dominant SRB mutations have the same suppression properties as a sca1 delta ::hisG strain. These results reveal a functional difference between positions two and five of the consensus CTD heptapeptide repeat. The ability of SCA1 and SRB mutant alleles to suppress CTD truncation mutations suggest that substitutions at position two, but not at position five, cause a defect in RNA polymerase II function similar to that introduced by CTD truncation.
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PMID:Suppression analysis reveals a functional difference between the serines in positions two and five in the consensus sequence of the C-terminal domain of yeast RNA polymerase II. 872 17

We have used supercoiled DNA templates in this study to demonstrate that transcription in vitro from the P1 and P2 promoters of the osmoresponsive proU operon of Escherichia coli is preferentially mediated by the sigma(s) and sigma70-bearing RNA polymerase holoenzymes, respectively. Addition of potassium glutamate resulted in the activation of transcription from both P1 and P2 and also led to a pronounced enhancement of sigma(s) selectivity at the P1 promoter. Transcription from P2, and to a lesser extent from P1, was inhibited by the nucleoid protein H-NS but only in the absence of potassium glutamate. This study validates the existence of dual promoters with dual specificities for proU transcription. Our results also support the proposals that potassium, which is known to accumulate in cells grown at high osmolarity, is at least partially responsible for effecting the in vivo induction of proU transcription and that it does so through two mechanisms, directly by the activation of RNA polymerase and indirectly by the relief of repression imposed by H-NS.
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PMID:Effects of H-NS and potassium glutamate on sigmaS- and sigma70-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli. 876 46

It is now well established that the sigma S subunit of RNA polymerase is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-inducible genes in Escherichia coli. In this review, more recent findings will be summarized that demonstrate that sigma S also acts as a global regulator for the osmotic control of gene expression, and actually does so in exponentially growing cells. Thus, many sigma S-dependent genes are induced during entry into stationary phase as well as in response to osmotic upshift. K+ glutamate, which accumulates in hyperosmotically stressed cells, seems to specifically stimulate the activity of sigma S-containing RNA polymerase at sigma S-dependent promoters. Moreover, osmotic upshift results in an elevated cellular sigma S level similar to that observed in stationary-phase cells. This increase is the result of a stimulation of rpoS translation as well as an inhibition of the turnover of sigma S, which in exponentially growing non-stressed cells is a highly unstable protein. Whereas the RNA-binding protein HF-I, previously known as a host factor for the replication of phage Q beta RNA, is essential for rpoS translation, the recently discovered response regulator RssB, and ClpXP protease, have been shown to be required for sigma S degradation. The finding that the histone-like protein H-NS is also involved in the control of rpoS translation and sigma S turnover, sheds new light on the function of this protein in osmoregulation. Finally, preliminary evidence suggests that additional stresses, such as heat shock and acid shock, also result in increased cellular sigma S levels in exponentially growing cells. Taken together, sigma S function is clearly not confined to stationary phase. Rather, sigma S may be regarded as a sigma factor associated with general stress conditions.
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PMID:Back to log phase: sigma S as a global regulator in the osmotic control of gene expression in Escherichia coli. 888 60

The Bradyrhizobium japonicum acnA gene encoding the tricarboxylic acid cycle enzyme aconitase was cloned and characterized. The gene was mapped immediately upstream of the cytochrome c biogenesis gene cycV and found to be transcribed in the opposite direction. The nucleotide sequence of acnA was determined; the derived amino acid sequence shared a significant similarity with bacterial aconitases and with the human iron-responsive-element-binding protein. The level of expression of the acnA gene under aerobic growth conditions was 10-fold higher than that under anaerobic conditions. The start of transcription was mapped by primer extension experiments, and the putative promoter was found to contain a typical -10 but no -35 consensus sequence for a sigma70-type RNA polymerase. A 5' deletion removing all but 19 nucleotides upstream of the start of transcription completely abolished gene expression. An acnA mutant was constructed by gene disruption, and the mutant phenotype was characterized. Growth of the mutant was severely affected and could not be corrected by the addition of glutamate as a supplement. Although aconitase activity in free-living cells was decreased by more than 70%, the ability of the mutant to establish an effective root nodule symbiosis with soybean plants was not affected. This suggested either the existence of a second aconitase or the compensation for the mutant defect by symbiosis-specific metabolites synthesized in the root nodules.
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PMID:The Bradyrhizobium japonicum aconitase gene (acnA) is important for free-living growth but not for an effective root nodule symbiosis. 889 15

The biosynthesis of the hemes, chlorophylls, corrins and other tetrapyrroles begins with the synthesis of 5-aminolevulinic acid (ALA). The pathway is highly conserved except for the synthesis of ALA which is derived from glycine and succinyl CoA (C4) in most eukaryotes and from glutamate (C5) in most bacteria and in green plants. In C5, glutamyl-tRNA synthetase (GTS) converts glutamate to glutamyl-tRNA (glu-tRNA), which is reduced by glutamyl-tRNA reductase (GTR) to glutamyl-1-semialdehyde (GSA), which is converted by aminotransferase (GSA-AT) to ALA. Since GTS is also involved in protein synthesis and GSA can be converted to ALA non-enzymatically, it is highly probable that control of ALA synthesis and thus of the whole pathway resides in the GTR step. In Escherichia coli, GTR is the gene product of hemA. BL21(DE3), a protease-deficient strain which contains the T7 RNA polymerase gene in front of a lac promoter, was transformed with a pET14b-based vector, pWC01, harboring hemA in front of a T7 promoter and ORF1 which is transcribed in the opposite direction. The transformed strain, WC1201, secreted ALA and porphyrins into the medium. Induction of expression of hemA by WC1201 was optimized for concentration of inducer (IPTG, 5 mM), temperature (37 degrees C), presence of betaine and sorbitol (no change) and time of induction (2h). GTR was observable as a 46 kDa band by Brilliant blue G staining of SDS-PAGE gels. Sonicates of the induction mixture exhibited strong ALA synthesis activity which was enhanced by tRNAglu. Most of the activity was in the supernatant of the sonicate indicating that GTR is a soluble enzyme. The induced strain had more GTS activity than the uninduced strain which had more GTS activity than its parent wild-type strain. Autoradiography on native gradient PAGE showed that GTR expressed in vivo by induction of WC1201 had a molecular weight of approx. 117 kDa. Gel filtration of the induced sonicate showed a peak of enzymatic activity at about 126 kDa. When pET14b- or pUC19-based plasmids harboring hemA and ORF1, or importantly, a pUC19-based plasmid harboring only hemA and not ORF1, were expressed in an in vitro transcription-translation system, native gradient PAGE showed a product with a molecular weight of approximately 175 kDA. This expression was higher in the presence of tRNAglu. When the 117 kDa and 175 kDa proteins were excised from their native gels respectively, and run on SDS PAGE, autoradiography showed bands at 46 kDa. We conclude that GTR is present in both high molecular weight species. Since overexpression of hemA from pET14b-based plasmids is associated with increased glutamyl-tRNA synthetase activity, the 175 kDa species may represent different complexes of GTR, GTS and glutamyl-tRNA as observed in Chlamydomonas and the 117-126 kDa species may be an dimer of GTR associated with glu-tRNA or a complex of GTR, GTS and glu-tRNA. These possibilities are being investigated.
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PMID:Expression of glutamyl-tRNA reductase in Escherichia coli. 895 Jan 86

Using oligonucleotide primers, we have amplified and sequenced the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors from the brain of 17-day-old chick embryos. Both flip and flop isoforms of each of these glutamate receptors (GluR) were identified and cloned. Nucleotide comparisons showed that the two isoforms for each chick receptor subtype were 71-78% identical, whereas homologous chick and rat isoforms were 94-98% identical. Reverse transcriptase-polymerase chain reaction and restriction enzyme analysis were employed to identify regional variation in flip and flop levels of each AMPA receptor. Flip isoforms of GluR 1-3 predominated in forebrain, while flop variants of GluR 1-4 were more prevalent in the cerebellum. This differential regional expression suggests that alternative splicing of AMPA receptor subunits contributes importantly to synaptic diversity in chick central nervous systems.
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PMID:Flip and flop isoforms of chick brain AMPA receptor subunits: cloning and analysis of expression patterns. 898 52


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