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 DNA-protein contact sites in the ara regulatory region, which contains the promoters for araBAD and araC, have been determined for araC protein, the cyclic AMP-binding protein, and RNA polymerase, by using the methylation protection and DNase I protection methods. The functional significance of binding was assessed by correlating the state of occupancy of these sites with promoter activity in transcription initiation. Our results suggest that the basis for araC autoregulation is that araC protein, in either its activator (P2) or repressor (P1) form, acts as a repressor for araC, by binding to the RNA polymerase attachment site at the araC promoter. We also found that the araC and araBAD promoters share a common site of positive control by the cyclic AMP-binding protein, located 90 bases from the araBAD and 60 bases from the araC transcriptional start points. A model for the mechanism of regulation of araBAD and araC expression by the catabolite gene-activator protein, P1, and Pe is proposed. An earlier model proposed by Ogden et al. [Ogden S., Haggerty, D., Stoner, C. M., Kolodrubetz, D. & Schleif, R. (1980) Proc. Natl. Acad. Sci, USA 77, 3346-3350] is discussed in the light of the data presented in this paper.
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PMID:Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli. 626 69

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

Superhelical pBR 322 derivatives have been relaxed by eukaryotic topoisomerase I in the presence or in the absence of E. coli cyclic AMP receptor protein (CRP) and of cyclic AMP (cAMP). CRP alone, or cAMP alone do not affect the average linking number of the distribution of the relaxed topoisomers. Hence, they do not unwind the template. In the presence of cAMP, CRP induces a small unwinding. The extent of this unwinding is barely modified when the relaxation is carried out on a similar vector plasmid where the CRP binding site of the lac or of the gal operon has been inserted. Under these conditions, we checked that CRP occupies the lactose control site and that upon addition of RNA polymerase, the corresponding promoter is readily activated. These findings are difficult to reconcile with the proposal that activation of these promoters results from the binding of the CRP-cAMP complex to left-handed DNA sequences.
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PMID:Is DNA unwound by the cyclic AMP receptor protein? 627 15

Restriction fragments containing the region preceding the tryptophanase structural gene, tnaA, were used as templates for in vitro transcription experiments. A transcription initiation site was detected that was dependent on the catabolite gene activator protein (CAP) plus cyclic AMP (cAMP). The mRNA produced in vitro was fingerprinted, and the nucleotide at which transcription was initiated was localized to the vicinity of two guanine residues 316 and 318 base pairs upstream of tnaA. A region exhibiting extensive difold symmetry and homology to the CAP binding site adjacent to the lactose operon promoter exists approximately 60 base pairs preceding the site of transcription initiation. Two HinfI restriction sites are located in this region. Restriction enzyme cleavage at these sites was prevented when DNA containing the promoter region was preincubated with CAP and cAMP. RNA polymerase was incapable of protecting these sites against this cleavage. CAP and cAMP addition did not protect against cleavage at a DdeI restriction site located in the -20 region of the promoter. RNA polymerase did protect against DdeI cleavage but only in the presence of CAP and cAMP. Thus, transcription initiation at the tryptophanase promoter involves cAMP-dependent, CAP-facilitated binding of RNA polymerase to the DNA.
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PMID:Transcription initiation at the tryptophanase promoter of Escherichia coli K-12. 628 18

The binding stoichiometries of the complexes formed when the E. coli cyclic AMP receptor protein (CAP) binds to 203 bp lac promoter-operator restriction fragments have been determined. Under quantitative binding conditions, a single dimer of CAP occupies each of two sites in the promoter. Different electrophoretic mobilities are observed for 1:1 complexes formed with L8-UV5 mutant, L305 mutant, and wild type promoter fragments, indicating sequence-specific structural differences between the complexes. The differences in gel mobility between L8-UV5 and wild type complexes disappear when the promoter fragments are cleaved with Hpa II restriction endonuclease. Models in which CAP alters DNA conformation or in which CAP forms a transient intramolecular bridge between two domains of a DNA molecule could account for these observations. The selective binding of RNA polymerase to CAP-promoter complexes is demonstrated: the binding of a single CAP dimer to the promoter is sufficient to stimulate subsequent polymerase binding. Functional CAP molecules are not released from the promoter on polymerase binding.
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PMID:CAP and RNA polymerase interactions with the lac promoter: binding stoichiometry and long range effects. 630 61

Mutations in the pts genes (which code for the enzyme I and HPr protein - the general components of the phosphoenolypyruvate-dependent phosphotransferase system) lead to decreases in enzyme-inducible synthesis at the level of transcription. The intracellular content of cyclic AMP in the ptsIH mutant was severely diminished, while the ptsH bacteria contain the same amounts of this nucleotide as the wild-type cells. Nevertheless expression of the lac operon was diminished in the ptsH as well as in the ptsIH mutant. The exogenous cyclic AMP did not prevent repression of beta-galactosidase synthesis in a delta cya ptsI mutant in a wide range of concentrations in the growth medium (from 0.05 mM to 5 mM). The combination of ptsI or ptsH mutations with rpoC1 (synthesis of thermosensitive beta' subunit of RNA polymerase) leads to greater disturbance of beta-galactosidase production at the nonpermissive temperature than demonstrated in the pts+ rpoC1 strain. The stimulatory effect of exogenous cyclic AMP was more pronounced in pts rpoC1 than in pts+ rpoC1 bacteria. The data presented confirm the hypothesis that pts mutations alter the function of CRP in initiation of transcription.
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PMID:Effect of ptsI and ptsH mutations on initiation of transcription of the Escherichia coli lactose operon. 630 97

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

DNase I footprinting experiments demonstrated that CAP, the cyclic AMP receptor protein of Escherichia coli, binds around position -70 at the promoter of malT, the positive regulator gene of the maltose regulon. The binding of CAP in the presence of cyclic AMP favored the subsequent specific binding of RNA polymerase. Initiation of malT transcription in vitro displayed an absolute requirement for CAP at all tested RNA polymerase concentrations. However this was not the case with a mutant promoter (malTp1), which leads to CAP-independent malT expression in vivo. In that case an effect of CAP was seen only at the lower concentrations of RNA polymerase. These results, which suggest that CAP stimulates malT expression by promoting the binding of polymerase to the promoter, are compared with those obtained in other systems.
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PMID:Action of CAP on the malT promoter in vitro. 631 76

Procedures for the synthesis, purification, and characterization of beta, gamma-peroxy analogues of the eight common ribo- and deoxyribonucleoside triphosphates have been developed. Although adenosine 5'-(beta, gamma-peroxytriphosphate) was stable to conditions in most biochemical systems, incubation of a solution of the analogue at 100 degrees C led to formation of AMP and ATP, as well as ADP. NAD+ pyrophosphorylase was the only enzyme among 13 tested for which adenosine 5'-(beta, gamma-peroxytriphosphate) was a good substrate, but the analogue was an effective inhibitor for a number of kinases. The peroxy compounds tested inactive with Escherichia coli RNA polymerase and DNA polymerase I, as well as with wheat germ RNA polymerase II.
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PMID:Nucleoside 5'-(beta, gamma-peroxytriphosphates). 631 17

Nuclear protein kinases include enzymes that transfer the gamma-phosphate of ATP to serine, threonine, lysine or histidine in proteins. Nuclear kinases with a preference for basic proteins are known as histone kinases; those preferring acidic protein substrates are casein kinases. Histone kinases include both cyclic AMP-independent protein kinases and cyclic AMP-dependent protein kinases. The best-characterized cyclic AMP-independent nuclear protein kinase is associated with cell proliferation and is activated (or transported to the nucleus) in G2 phase of the cell cycle. It phosphorylates specific serine and threonine residues in the non globular domains of histone H1 and appears to promote chromosome condensation. The cyclic AMP-dependent protein kinase has unknown nuclear function(s), although it may be translocated from cytoplasm to nucleus in response to specific hormonal stimuli which are also associated with changes in transcriptional activity. There is a massive peak of nuclear cyclic AMP-dependent protein kinase activity in G2 phase of the cell cycle. Nuclear casein kinases are apparently very heterogeneous. Two of these enzymes have been purified to homogeneity. They phosphorylate non-histone chromosomal proteins, including RNA polymerase and ornithine decarboxylase. Phosphorylated ornithine decarboxylase is inactive enzymatically but, in Physarum, it binds to the rDNA minichromosome and stimulates rRNA transcription. Kinases forming phosphoramidate bonds occur in a variety of rat tissues and form phosphohistide in histone H4 and phospholysine in histone H1.
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PMID:Nuclear protein kinases. 632 62


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