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)

Micrococcal nuclease (MNase) is used to probe the structure of transcription and repression complexes at the lac regulatory region in vitro. Both the lac operator, 01, and the pseudo-operator, 03, are found to be protected from MNase digestion by the lac repressor on supercoiled DNA, and hypersensitive sites appear on both strands around nucleotide (nt) -26 between 01 and 03. This hyperreactive site is coincident with the site of the DNA kink shown previously to form within a loop caused by simultaneous repressor binding to 01 and 03. MNase hypersites are also observed both upstream from cAMP receptor protein (CRP) and downstream from bound RNA polymerase in open promoter complexes. In both open and closed complexes the binding of polymerase partially protects the backbone from MNase attack. Catabolite activator protein is shown to be required for both closed and open complex formation. Taken together with previous footprinting data, the results suggest that lac transcription complexes involve DNA bent towards a protein core consisting of RNA polymerase and catabolite activator protein.
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PMID:Micrococcal nuclease as a probe for bound and distorted DNA in lac transcription and repression complexes. 266 75

Expression of ornithine decarboxylase is regulated by a variety of hormonal and other stimuli in rat cells and tissues. To study this phenomenon at the molecular level, we isolated and sequenced a cDNA-encoding rat ornithine decarboxylase and deduced its amino acid sequence. The cDNA clone was used to isolate a clone from a rat genomic library which contained the sequence of the entire rat ornithine decarboxylase gene. The gene comprised 12 exons and 11 introns and spanned 7.7 kilobases. Two polyadenylation signals (AATAAA) were located 310 and 697 base pairs 3' to the translational termination codon and were responsible for the occurrence of two hybridizing mRNA species in Northern blots of rat cells and tissues. S1 nuclease mapping suggested that there were multiple transcriptional start sites; the major one appeared to be located 2269 base pairs of genomic sequence 5' to the ATG translational initiation site, representing 274 bases of mRNA. Several potential regulatory elements were identified in the 5'-promoter regions or in the first intron: a TATA box, GC boxes, AP-1 and AP-2 binding sites, a cAMP-responsive element, a glucocorticoid regulatory element, and RNA polymerase III promoter sequences. The 5'-noncoding region of the mRNA was extremely rich in G + C; secondary structure predictions suggested that almost this entire region could form stable secondary structures, with an overall free energy of formation (delta G) of -114 kcal/mol. The potential regulatory elements identified in both the promoter region of the gene and the 5'-untranslated region of the mRNA may be involved in the complex regulation of rat ornithine decarboxylase expression.
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PMID:Rat ornithine decarboxylase gene. Nucleotide sequence, potential regulatory elements, and comparison to the mouse gene. 272 15

The effects of corticotropin releasing factor (CRF) on pro-opiomelanocortin (POMC) gene expression were investigated in an anterior pituitary corticotrophic tumor cell line, AtT-20/D16-16. The results of mRNA dot blot hybridization assays suggested that CRF, at a concentration of 10(-7) M, positively regulates the expression of the POMC gene in AtT-20 cells in a concentration-dependent fashion. Evaluation of the time course of this effect indicated that CRF had a biphasic mode of action. CRF and alpha-amanitin (inhibitor of RNA polymerase II activity) were also found to affect POMC mRNA levels in a concentration-dependent fashion. Eight-bromo-cyclic adenosine monophosphate (8-Br-cAMP) produced biphasic effects on POMC mRNA levels, supporting evidence of a role for cAMP as a second messenger in the regulation of POMC gene expression. It was also found that alpha-amanitin negatively regulated basal and CRF-stimulated POMC mRNA levels at both the 2 hr and 24 hr time periods, supporting evidence for positive regulation of POMC by CRF at the transcriptional level.
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PMID:CRF and cAMP regulation of POMC gene expression in corticotrophic tumor cells. 282 45

The murine urokinase-type plasminogen activator (uPA) gene has been isolated from a BALB/c liver DNA cosmid library and its nucleotide sequence established. The gene is organized into 11 exons comprising 34.7% of the 6710 base pair (bp) region spanning the interval between the presumed transcription initiation and polyadenylation sites. The transcription initiation site is flanked by common RNA polymerase II promoter elements, including a TATA box and a potential transcription factor Sp1 binding site. A large polypurine tract of the structure (AG)22(AGGG)16(AG)28 is located 79 bp upstream of the 5'-terminus. It was highly sensitive to the single-strand-specific nuclease S1, suggesting a non-B-DNA conformation of unknown significance. Consistent with the well-documented influence of adenosine cyclic 3',5'-phosphate (cAMP) on uPA gene expression, there is a dodecanucleotide homologous to proposed regulatory sequences identified in other cAMP-modulated genes. Comparison of the murine uPA gene to the previously described porcine and human uPA genes revealed an unusually high degree of evolutionary (interspecies) sequence conservation that was not limited to exons but included introns and flanking sequences as well.
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PMID:The murine urokinase-type plasminogen activator gene. 283 40

In Escherichia coli, the transcription of the malT gene is activated by the complex formed between cAMP and its receptor protein, CRP. Kinetics of formation of polyribonucleotide products from the corresponding promoter were studied in vitro by two sets of techniques, abortive initiation assays and run-off experiments. The first type of assay indicated that open complexes were formed at malT with an equivalent efficiency, and at comparable rates, whether CRP-cAMP was present or not. Secondary effects due to the activating complex were observed (increased stability of the open complex, elimination of a weaker binding site for the enzyme, improved Michaelis constants of RNA polymerase for the substrates of the assay, UTP in particular). But, primarily, CRP-cAMP did not exert a significant role in the rate of formation of the initiation complex. In contrast, run-off assays showed that the yield of the full-length transcripts was markedly enhanced by prior incubation of the DNA fragment with CRP-cAMP. Both in the presence and in the absence of activator, the rate-limiting step for this process was markedly slower than the formation of the initial open complex. Short oligonucleotides (n less than 9), probably arising from a recycling process, were found when the initiation complex was formed in the absence of CRP-cAMP. They were abolished by prior incubation with the activator. Unexpectedly, CRP-cAMP appears to favour the escape of RNA polymerase from the initiation complex at this promoter.
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PMID:A new target for CRP action at the malT promoter. 283 58

Four cAMP-independent receptor protein mutants (designated CRP* mutants) isolated previously are able to activate in vivo gene transcription in the absence of cAMP and their activity can be enhanced by cAMP or cGMP. One of the four mutant proteins, CRP*598 (Arg-142 to His, Ala-144 to Thr), has been characterized with regard to its conformational properties and ability to bind to and support abortive initiation from the lac promoter. In the absence of cGMP, CRP*598 shows a more open conformation than CRP, as indicated by its sensitivity to proteolytic attack and 5,5'-dithiobis(2-nitrobenzoic acid)-mediated subunit crosslinking. Binding of wild-type CRP to its site on the lac promoter and activation of abortive initiation by RNA polymerase on this promoter are effected by cAMP but not by cGMP. CRP*598 can activate lacP+-directed abortive initiation in the presence of cAMP and less efficiently in the presence of cGMP or in the absence of cyclic nucleotide. DNase I protection ("foot-printing") indicates that cAMP-CRP* binds to its site on the lac promoter whereas unliganded CRP* and cGMP-CRP* form a stable complex with the [32P]lacP+ fragment only in the presence of RNA polymerase, showing cooperative binding of two heterologous proteins. This cooperative binding provides strong evidence for a contact between CRP and RNA polymerase for activation of transcription. Although cGMP binds to CRP, it cannot replace cAMP in effecting the requisite conformational transition necessary for site-specific promoter binding. In contrast, the weakly active unliganded CRP*598 can be shifted to a functional state not only by cAMP but also by cGMP and RNA polymerase.
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PMID:Cooperative DNA binding of heterologous proteins: evidence for contact between the cyclic AMP receptor protein and RNA polymerase. 283 57

We have isolated a new class of mutations in rpoD, the gene encoding the sigma 70 subunit of Escherichia coli RNA polymerase, that alter the transcription initiation properties of RNA polymerase holoenzyme. The rpoD(Lac) mutations increase expression of the lac operon in the absence of CAP-cAMP, allowing a strain lacking adenyl cyclase to grow on lactose. Four of the six alleles isolated have three- to fivefold increases in the amount of lac mRNA and beta-galactosidase per cell. We show that these four mutations increase transcription initiation from the same promoter used by wild-type RNA polymerase. The mutations were mapped and sequenced. One mutation occurs in the codon for amino acid 389 of the sigma 70 polypeptide. The remaining five mutations are clustered, affecting residues 570, 571 and 575. These five mutations are within or near a proposed helix-turn-helix motif in the C terminus of sigma 70.
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PMID:Mutations in rpoD, the gene encoding the sigma 70 subunit of Escherichia coli RNA polymerase, that increase expression of the lac operon in the absence of CAP-cAMP. 284 53

Expression of the melR gene is required for melibiose-dependent stimulation of transcription initiation at the promoter of the melAB operon. Using the S1 nuclease method we have located the melR transcription start point. Transcription from the melR promoter is dependent on cAMP-CRP: specific nucleotide sequences downstream of bp -59 with respect to the melR transcription start are sufficient for full promoter activity. Nucleotide sequence homologies suggest that the cAMP-CRP binding site is located from bp -52 to -31, in exactly the same position as at the galP1 promoter. Using DNase I footprinting we show that cAMP-CRP and RNA polymerase together bind tightly to the melR promoter sequence, creating a strong footprint from bp -70 to +20. Alone, cAMP-CRP binding is hardly detectable, whereas RNA polymerase alone creates a weak footprint centred around the -10 hexamer sequence. When the melR gene is expressed from a cAMP-CRP-independent promoter, melibiose-dependent transcription from the melAB promoter becomes independent of cAMP-CRP, showing that the melR promoter is the primary site of control by cAMP-CRP in the mel regulon.
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PMID:Transcription from the Escherichia coli melR promoter is dependent on the cyclic AMP receptor protein. 285 97

Specialized lambda-transducing phages that carry the Escherichia coli genes ptsH, ptsI, crr, cysM, and cysA have been isolated, and the genes were subcloned in plasmid pBR322. Subcloning and restriction mapping data gave the following clockwise order of genes located at about 52 min on the E. coli genetic map: lig, cysK, ptsH, ptsI, crr, cysM, cysA. The nucleotide sequences of ptsH, ptsI, and crr and the corresponding flanking regions have been determined. These genes encode three cytoplasmic proteins of the phosphoenol-pyruvate:glycose phosphotransferase system: HPr, Enzyme I, and IIIGlc, respectively. The deduced amino acid sequences are consistent with amino acid composition and Edman degradation analyses obtained with the purified proteins. The calculated subunit molecular weight values (9,109 for HPr, 63,489 for Enzyme I, and 18,099 for IIIGlc) also agree well with values obtained with the proteins. Results of gamma delta-transposon insertional studies provided definitive evidence that IIIGlc is the gene product of crr, and therefore that IIIGlc plays a critical role in regulating the metabolism and uptake of certain non-PTS sugars (see accompanying papers: Mitchell, W.J., Saffen, D.W., and Roseman, S. (1987) J. Biol. Chem. 16254-16260; Misko, T.P., Mitchell, W.J., Meadow, N.D., and Roseman, S. (1987) J. Biol. Chem. 16261-16266). The gamma delta transposon studies also suggest that crr is transcribed from an independent promoter located within the ptsI gene. Putative regulatory sequence features include a catabolite gene activator protein-cAMP-binding site and two regions of 2-fold rotational symmetry adjacent to the potential promoter upstream from the HPr structural gene, several ribosome-binding sites, and a rho-independent RNA polymerase termination site downstream from crr. In addition, the ptsI gene contains two highly conserved direct repeats. The significance of these sequence features is discussed with respect to possible multiple forms of pts regulation.
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PMID:Sugar transport by the bacterial phosphotransferase system. Molecular cloning and structural analysis of the Escherichia coli ptsH, ptsI, and crr genes. 296 Jun 75

The regulatory region of the Escherichia coli cya gene was analyzed by using S1 nuclease mapping and in vitro transcription experiments. The cya gene was transcribed, both in vivo and in vitro, from one major promoter (P2) and two weak promoters (P1 and P1') that are located about 200 base pairs upstream of P2. The transcription from P2 was specifically inhibited by cAMP-CRP (cAMP receptor protein) in vitro. This regulatory mechanism was shown to be physiologically relevant through quantitative analyses of the cya mRNA in intact cells by S1 and dot blot assays. DNase I protection experiments revealed that cAMP-CRP binds to the cya DNA region between +11 and -20, in which a consensus CRP binding sequence is present. Moreover, it was found that cAMP-CRP alters the binding of RNA polymerase to the promoter region, thus inhibiting the transcription of the cya gene.
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PMID:Transcription of the Escherichia coli adenylate cyclase gene is negatively regulated by cAMP-cAMP receptor protein. 298 47


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