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 influence of cAMP-dependent pig brain protein kinase and its subunits on transcription in vitro was studied. The increase in the template activity of chromatin isolated from the nuclei after treatment with the catalytic subunit was observed. The regulatory subunit of protein kinase was found to increase the number of binding sites for RNA polymerase on chromatin. The cAMP-dependent pig brain protein kinase was found to phosphorylate the sigma-factor of Escherichia coli RNA polymerase. This phosphorylation led to the increase of the RNA polymerase activity on phage lambda DNA. The nuclear translocation of the protein kinase and its subunits was shown to take place. In the cells with a low cAMP level (SV40 3T3) the transfer of the regulatory subunit to the nucleus was not detected. Only upon addition of cAMP and a subsequent formation of the cAMP-regulatory subunit complex, nuclear translocation was observed in these cells. The dependence of nuclear translocation of the cAMP-dependent protein kinase on cAMP level in the cell is proposed.
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PMID:[Nuclear translocation and effect of cAMP-dependent protein kinase on transcription]. 626 Feb 40

The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified. This gel method is applied to the study of the E. coli lactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promotor, but not with a CAP-insensitive mutant. Formation of a stable "open" or "melted-in" complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.
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PMID:A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. 626 71

The preincubation of a DNA with E. coli RNA polymerase provides its partial protection against the HindII, BspRI and AluI cleavage giving possibility to determine the location of RNA polymerase tight-binding sites. Using this approach about 16 RNA polymerase tight-binding sites were detected on replicative form of phiX174 phage DNA. The protection degree of each of these sites depended on the preincubation conditions. Some of the protected sites hit the known phiX174 promoters and rho-dependent terminators and the other were distributed along the whole phiX174 DNA molecule. Many of them could be considered as potential promoters because they contain all the necessary elements specifying the real promoter sequences. At least some of the intrinsic promoter elements could be observed next to the rest of protected sites. One of the protected sites (R6b/l) is located in phiX174 DNA region which is very similar to the cAMP-CRP-controlled promoter sequences. It was confirmed that phiX174 DNA has two B promoters positioned by Sanger on the phiX174 nucleotide map, according to our data obtained by RNA polymerase protection experiments along with RNA product analysis of the R8 DNA fragment transcription in vitro.
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PMID:[Protection of segments of replicative form I of phiX174 phage DNA recognized by HindII, BspRI, and AluI restrictases by Escherichia coli RNA-polymerase]. 627 99

The restriction fragments carrying the region preceding the Escherichia coli crp structural gene were transcribed. The 5' end of the crp mRNA was determined by RNAase partial digestion and S1 digestion methods. Thus the crp gene has been shown to possess a 167 bp leader. CRP-cAMP specifically prevents the crp transcription. In other words, the crp gene is regulated autogenously. DNAase foot-printing studies indicated that CRP-cAMP binds to the crp gene at positions +26 to +67. This region exhibits a striking sequence homology to the CRP-binding sites in other genes. CRP and RNA polymerase bind to the crp regulatory region simultaneously. These results suggest a different mechanism for transcriptional repression of the crp gene by CRP-cAMP from that of a typical operator-repressor model.
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PMID:Autoregulation of the Escherichia coli crp gene: CRP is a transcriptional repressor for its own gene. 629 82

The catabolite activator protein (CAP) of Escherichia coli, complexed with cAMP, is required for efficient initiation of transcription from the galactose P1 promoter (start site at +1) but not from the overlapping P2 promoter (start site at -5) [Musso, R. E., DiLauro, R., Adhya, S. & deCrombrugghe, B. (1977) Cell 12, 847-854]. We investigated the interactions between CAP/cAMP and the gal promoter region in the presence of RNA polymerase. DNase I protection experiments of gal promoter restriction fragments revealed that CAP/cAMP protects the DNA from digestion between positions -50 and -25 and that RNA polymerase protects it from -35 to +10; however, gal DNA in the presence of both CAP/cAMP and RNA polymerase is protected from DNase I digestion between positions -68 and +15. Results of exonuclease III protection experiments show that RNA polymerase alone protects the gal DNA from -30 to +15; when both CAP/cAMP and RNA polymerase are present in the reaction, protection is afforded from -65 to +20. We directly quantified the amount of cAMP and CAP bound to gal promoter DNA in the presence of RNA polymerase by selectively pelleting the ternary complexes (CAP/cAMP-RNA polymerase-gal promoter DNA) in a Beckman Airfuge. We found two CAP molecules specifically bound to the gal promoter, although only one cAMP molecule was found in the complex at low cAMP concentrations (but sufficient to support P1 transcription). Thus, both the DNA protection experiments and the centrifugation results indicate that RNA polymerase induces the binding of a second CAP molecule to the gal promoter in forming stable initiation complexes. It appears that the second CAP molecule is needed to stimulate initiation from the P1 promoter; this may be involved in regulating the relative rates at which transcription begins from the two gal start sites.
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PMID:Two catabolite activator protein molecules bind to the galactose promoter region of Escherichia coli in the presence of RNA polymerase. 630 Aug 59

We have examined the interaction site on gal DNA for the cyclic AMP receptor protein and RNA polymerase when both are present together to form a stable initiation complex at the P1 gal promoter. Substitution of the bases to the left of -60 by unrelated DNA sequences does not change the cyclic AMP concentration dependency for in vitro transcription at P1 and inhibition of P2. Although the presence of some DNA to the left of -60 appears to be needed for efficient in vitro transcription at P1, the gal sequence to the left of -60 does not provide any specific interactions for transcription initiation at P1. Similarly, efficient in vitro transcription from P2 also requires non-specific DNA sequences to the left of -60. We have also examined which bases were protected by RNA polymerase and CRP together from the action of DNAase and dimethylsulfate. Some of the interactions that take place when cAMP-CRP alone interacts with gal DNA appear to be preserved in the cAMP-CRP-RNA polymerase-gal DNA complex, suggesting that CRP occupies the same site in the DNA when it is alone or together with RNA polymerase. Our results suggest that the formation of an open complex at different promoters can result from different interaction patterns between RNA polymerase and promoter DNA.
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PMID:Interactions of RNA polymerase and the cyclic AMP receptor protein on DNA of the E. coli galactose operon. 630 75

Using DNase footprinting and transcription assays in vitro we have probed the effect of the cAMP-cAMP receptor protein complex (cAMP-CRP) on the positioning of RNA polymerase and on the location of the transcription start point at the Escherichia coli gal and lac operon regulatory regions. In both cases, RNA polymerase can form two alternative complexes which promote transcription from two different start points, S1 and S2: pre-incubation of promoter DNA with cAMP-CRP results in a shift of the transcription start from S2 to S1 and in an increase in the rate of open complex formation. Moreover, the rate of formation of each heparin-resistant complex parallels the establishment of the corresponding footprint, showing that the stable binding corresponds to open complex formation. We show that, in the case of gal, RNA polymerase, which is bound so as to transcribe from S2, cannot be diverted to S1 by subsequent addition of cAMP-CRP. In contrast, in the case of lac, when cAMP-CRP is added after RNA polymerase, complexes which initiate transcription at S2 are rapidly converted to complexes which initiate at S1. Finally, we present data which suggest that protein-protein interactions are essential for CRP-induced activation at both the lac and gal promoters.
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PMID:On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions. 632 69

The control of androgen production by the Leydig cell is dependent upon the episodic secretion of hormone (LH), which is released from the anterior pituitary gland in pulses of high biological activity. This mode of episodic LH secretion supports steroidogenic enzyme activity in the testis through interaction with LH receptors and stimulation of the adenylate cyclase/protein kinase sequence, leading to phosphorylation of key intermediates in the steroid biosynthetic pathway. The plasma membrane events that are rapidly activated by the specific interaction of LH or hCG with Leydig cell receptors include increased binding of guanyl nucleotide, and stimulation of cAMP-independent, Ca2+-dependent phosphorylation of a 44,500 Mr protein, with the characteristics of the adenylate cyclase nucleotide regulatory unit. Hormonal activation of adenylate cyclase is affected by Ca2+ with the same concentration-dependence, suggesting that nucleotide-induced phosphorylation is related to activation of the catalytic cyclase unit. In addition to the characteristic increases in pregnenolone synthesis and androgen production, gonadotropin-stimulated Leydig cells show prominent changes in LH receptor content and steroidogenic activity that modify their subsequent responses to hormonal signals. Thus, after exposure to increased LH and hCG levels in vivo and in vitro, LH receptors show an initial transient increase (up-regulation) followed by a marked decrease (down-regulation) and a prolonged depletion of LH receptor sites. Large doses of hCG cause "early" (prior to pregnenolone) and "late" steroidogenic lesions (17 alpha-hydroxylase, 17-20 desmolase) that are independent of receptor loss. The early lesion is partly due to reduced activity of HMG CoA reductase, and is mainly attributable to the increased activity of an inhibitory protein factor that modulates the activity of cholesterol side chain cleavage enzyme in Leydig cell mitochondria. In contrast, the late steroidogenic lesion is related to the nuclear actions of E2 produced during hormonal action. After hCG stimulation, an increase in nuclear E2 binding was accompanied by an early rise of RNA polymerase activities within 45 min coincident with the maximal increases in circulating testosterone and estradiol levels. These events were followed by the emergence of an E2-induced protein of Mr 27,000 at 3-6 h, and by reduction in the activity of 17 alpha-hydroxylase/17-20 desmolase, and a decrease in microsomal cytochrome P-450.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Hormonal regulation of androgen production by the Leydig cell. 632 62

Two mutations that affect expression of the Salmonella typhimurium leu operon were investigated. leu operon DNA from these mutant strains was cloned, and nucleotide sequences of the leu control regions were determined. leu-500, which eliminates expression of all four leu genes simultaneously, is a point mutation in the -10 region of the leu promoter. leu-2012 is a point mutation within the -35 region of the leu promoter. leu-2012 suppressed leucine auxotrophy caused by leu-500 only when the medium contained a carbon source that does not cause catabolite repression. A cya mutation (adenylate cyclase deficiency) introduced into the leu-500 leu-2012 strain caused leu enzymes to be made only if cAMP was supplied exogenously. A leu-500 leu-2012 strain containing a crp mutation (cAMP receptor protein deficiency), on the other hand, could not make leu enzymes even in the presence of cAMP. In vitro transcription experiments demonstrated that the leu-2012 mutation created a new transcription initiation site. RNA polymerase utilized this site in vitro in the absence of added cAMP receptor protein and cAMP.
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PMID:Promoter mutation causing catabolite repression of the Salmonella typhimurium leucine operon. 632 52

An intriguing mechanism in regulating transcription initiation from the gal operon in Escherichia coli is described. Initiation from galP2, one of the two promoters of the E. coli galactose operon, is shown to be subject to promoter clearance control in responding to changes in UTP concentration. In vitro, RNA polymerase (RNAP) makes a large amount of nonproductive "stuttering" initiation products at the galP2 promoter at high concentrations of UTP and less of the stuttered products at low concentrations of UTP. Conversely, RNAP makes more productive initiation products at low UTP concentration than at high UTP concentration. The transcription factor cAMP.CRP complex which normally inhibits transcription from galP2 also represses the stuttering synthesis from galP2. When galactose is used as a sole carbon source and the internal UTP pools are adjusted externally, a cya mutant (in which galP2 is mainly responsible for the expression of the gal operon and galP1 activity is minimal) has a slower growth rate and lower expression of the gal operon at high UTP pools than at low UTP pools. Such an apparent correlation between the in vitro and in vivo results allows one to speculate that changes in UTP concentration can modulate the expression of the gal operon. The implication of a gal promoter being controlled by UTP is discussed.
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PMID:Slippage synthesis at the galP2 promoter of Escherichia coli and its regulation by UTP concentration and cAMP.cAMP receptor protein. 751 34


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