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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 Streptomyces galactose operon is transcribed from two independently regulated promoters: galP1, located at the 5' end of the operon and responsible for galactose-dependent transcription of the operon, and galP2, an internal constitutive promoter. We identified and partially separated two distinct transcribing activities involved in expression of this operon. Using RNA polymerase from Streptomyces lividans and Streptomyces coelicolor partially purified by chromatography on heparin-agarose and DNA-cellulose, we detected activities capable of initiating transcription in vitro specifically from either galP1 or galP2. Circumstantial evidence suggests that the activity for galP2 transcription is a holoenzyme species associated with the previously described sigma 28 protein (referred to here as sigma C). The galP1-transcribing activity is more difficult to evaluate. This activity may correspond to a holoenzyme species associated with sigma A (formerly sigma 35), although other possibilities are discussed. This would be the second reported example of a catabolite-controlled gene in Streptomyces species expressed from multiple promoters recognized by different holoenzyme forms. This may indicate that the involvement of RNA polymerase heterogeneity in gene expression in Streptomyces species is a more general strategy for regulation than the specialized gene expression seen in Escherichia coli.
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PMID:Two transcribing activities are involved in expression of the Streptomyces galactose operon. 292 Dec 38

Two overlapping promoters compete for RNA polymerase in the region that controls the expression of the galactose operon in Escherichia coli. Kinetics of open complex formation at P1 and P2 can be followed through the rate of formation of two specific abortive transcripts. The corresponding forward kinetic constants appear to be identical over a wide range of enzyme concentrations and temperatures, indicating that the two processes are strongly coupled. We propose a scheme accounting for our observations. In a first step, the competition between the two sites is a simple kinetic process, involving the "on" rate constants. In a second step, a slow reequilibration occurs, implicating the "off" rate constants and the conversion of one open complex to the other through a set of closed complexes. The first step is clearly affected when the complex between cyclic AMP and its receptor is bound at the activator site. An estimate of the various rate constants describing open complex formation at P1 and P2 is provided, as well as a qualitative description of the effect of the activator complex on these two pathways.
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PMID:Overlapping promoters and their control in Escherichia coli: the gal case. 301 Mar 19

The Escherichia coli galactose and lactose promoter regions have been studied by alkylation interference experiments. The data reveal those bases or phosphate groups which, when modified, prevent the binding of the catabolite activator protein (CAP) or RNA polymerase and hence are presumably in contact with the proteins. Interference contacts made by CAP at its primary binding sites at gal and lac are quite similar, indicating that CAP-cAMP uses the same mode of binding at these two operons. RNA polymerase, when bound in the presence of CAP-cAMP, exhibits contacts at the gal and lac P1-10 regions very much like those of the lac UV5 and T7 A3 promoters (Siebenlist, U., Simpson, R. B., and Gilbert, W. (1980) Cell 20, 269-281). CAP, therefore, does not detectably alter the structure of the open complex. The binding sites for CAP and RNA polymerase at lac, as deduced from interference experiments, do not overlap. However, at gal a CAP molecule is found much closer to the enzyme, and there is competition for a set of mutual contacts. These experiments thus reveal both similarities and differences in the mechanisms whereby CAP activates transcription at catabolite-sensitive operons.
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PMID:The binding of catabolite activator protein and RNA polymerase to the Escherichia coli galactose and lactose promoters probed by alkylation interference studies. 301 46

The molecular mechanisms whereby RNA polymerase, catabolite activator protein (CAP), and cyclic AMP (cAMP) participate in transcriptional regulation at the galactose operon have been probed by a variety of in vitro techniques. Interactions between purified proteins and promoter-containing DNA fragments were assayed by gel electrophoresis, by resistance to restriction endonuclease digestion, and by monitoring runoff transcripts. The data bear on the multiple functions that CAP performs in gal control. A CAP-cAMP complex can exclude RNA polymerase from one of the two overlapping promoter regions (P2), thereby targeting the enzyme to the other (P1); this process is markedly influenced by the cAMP level. In addition, a second CAP molecule is involved in a cooperative process, which, at low cAMP, is required for efficient formation of transcriptionally competent complexes at P1. This second CAP may serve to stabilize the 1:1:1 CAP-polymerase-gal DNA intermediate under physiological conditions, thus enhancing initiation from P1 relative to P2. Kinetic analysis reveals that the modest effect of CAP on the rate of P1 open complex formation can be resolved into about a 4-fold increase in the binding of RNA polymerase to the P1 region, plus a 1.5-fold elevation in the rate of isomerization of enzyme-promoter complexes to the open state.
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PMID:Role of a second catabolite activator protein molecule in controlling initiation of transcription at the galactose operon of Escherichia coli. 302 95

Two mutations are described, each of which renders the Pribnow box sequence of one of the two overlapping promoters of the Escherichia coli galactose operon identical to the consensus sequence TATAAT. Both double exchanges were specifically introduced into the original context by oligonucleotide-directed mutation construction. Each of the mutant promoters exhibits a greatly enhanced capacity to form stable complexes with RNA polymerase, as judged by nuclease protection experiments and by assaying shifts of electrophoretic mobility. On the other hand, the effect of the same mutations on the rates of transcription from the two gal promoters is strikingly different. Unexpectedly, when complexed with RNA polymerase, DNA fragments carrying one of the two double exchanges were found to differ from each other as well as from the corresponding wild-type fragment with respect to their electrophoretic mobilities. These observations are indicative of different three-dimensional structures of these complexes which may reflect different forms of DNA bending induced in these otherwise identical fragments by complex formation with RNA polymerase.
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PMID:Unusual properties of promoter-up mutations in the Escherichia coli galactose operon and evidence suggesting RNA polymerase-induced DNA bending. 303 93

The catabolite activator protein (CAP) binding sites of the Escherichia coli galactose and lactose operons were probed by hydroxyl radical footprinting. This method reveals each base that is protected by the bound protein. The patterns of protection seen for the primary CAP sites at gal and lac were virtually identical. In the presence of RNA polymerase the footprint of the second CAP molecule at gal was found to be very similar to those at the other two sites. This upstream site in gal align's perfectly with the lac CAP site with respect to the start of P1 transcription. Replacing most of the gal second CAP site DNA with heterologous sequences did not abolish binding although it became noticeably weaker. In vitro transcription studies of this hybrid gal promoter DNA further demonstrated the reduced affinity of the second CAP. These results are consistent with molecular models proposed for specific CAP binding and suggest that the second CAP at gal may be responsible for overall stimulation of transcription at this operon. Thus, in spite of differences in stoichiometry, the mechanisms of activation by CAP at gal and lac may be quite similar.
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PMID:Interactions of the catabolite activator protein (CAP) at the galactose and lactose promoters of Escherichia coli probed by hydroxyl radical footprinting. The second CAP molecule which binds at gal and the one CAP at lac may act to stimulate transcription in the same way. 304 Jul 1

We have constructed a yeast strain (UKY403) in which the sole histone H4 gene is under control of the GAL1 promoter. This allows the activation of H4 mRNA synthesis on galactose and its repression on glucose. UKY403 cells, pre-synchronized in G1 with alpha-mating factor, have been used to show that glucose treatment results in the loss of approximately half the chromosomal nucleosomes. This depletion is only partially reversible when the H4 gene is reactivated on galactose. It was found that the resultant lethality manifests itself first in S phase, the period of nucleosome assembly, but leads to highly synchronous arrest in G2 and a virtually complete block in chromosomal segregation. Histone H4-depleted chromatin was analyzed for its efficiency as a template for all three RNA polymerases. Using pulse-labeling, we find no evidence for altered transcription by RNA polymerase I (25S, 18S and 5.8S rRNAs) or RNA polymerase III (5S rRNA, tRNAs). Northern blot analysis was used to measure levels of RNA polymerase II transcripts. There was little effect on the activation or repression of the CUP1 chelatin gene. While there may be some decrease in the level of certain mRNAs (e.g. HIS4, ARG4) other message levels (HIS3, TRP1) show little change upon glucose repression. Therefore, nucleosome loss certainly does not have a general effect on transcription.
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PMID:Effects of histone H4 depletion on the cell cycle and transcription of Saccharomyces cerevisiae. 304 33

Starting with a DNA fragment containing the galactose operon P2 promoter, we made a series of deletions that progressively replaced DNA sequences upstream of the transcription startpoint and determined their effects on P2 activity. The results show that specific sequences upstream of -32 are not important. Removal of the sequence 5'-CACA-3' from -32 to -28 reduces P2 activity by 50%: longer deletions to -16 further reduce activity but do not remove the information specifying the transcription startpoint. DNA sequences between -32 and -16 at gal P2 assist the isomerization of RNA polymerase from closed to open complexes rather than contributing to the initial binding of RNA polymerase. The activity of gal P2 in the absence of -35 region sequences is dependent on the sequence TG just upstream of the -10 hexamer, TATACT: a mutation at -14 changing the TG sequence to TT totally inactivates P2. However, P2 activity can be restored if the consensus -35 region sequence TTGACA is cloned 17 bp upstream of the -10 hexamer. Thus, for transcription initiation, the -10 hexamer, TATACT, must 'cooperate' with upstream sequences that may be located either around -35 or -14.
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PMID:Functional analysis of different sequence elements in the Escherichia coli galactose operon P2 promoter. 328 31

We report in vitro studies of the interactions between purified E. coli RNA polymerase and DNA from the regulatory region of the E. coli galactose operon which carries a point mutation that simultaneously stops transcription initiation at the two normal start points, S1 and S2. In the presence of this point mutation, transcription initiates at a third start point 14/15 bp downstream of S1, showing that inactivation of the two normally active promoters, P1 and P2, unmasks a third weaker promoter, P3. Transcription initiation in the gal operon is normally regulated by the cyclic AMP receptor protein, CRP, that binds to the gal regulatory region and switches transcription from P2 to P1. With the point mutation, CRP binding switches transcription from P3 to P1, although the formation of transcriptionally competent complexes at P1 is very slow. The results are discussed with respect to the mechanism of transcription activation by the CRP factor and the similarities between the regulatory regions of the galactose and lactose operons.
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PMID:Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters. Interactions with RNA polymerase and the cyclic AMP receptor protein. 329 89

Administration of galactose into young rats within an early postnatal period led to alteration in activity of some enzymes involved in utilization of galactose (galactose-1-phosphaturidyl transferase, galactose-6-phosphate dehydrogenase etc) for a long period of the animals life. This stable alteration in activity of adaptive enzymes was characterized as the enzymatic imprinting. After administration of galactose into neonatal animals synthesis of RNA, matrix activity of chromatin, activities of DNA-dependent RNA polymerase and RNA-dependent DNA polymerase were shown to increase in liver tissue of these animals. These alterations are considered as a possible basis for the stable alterations in the genes expression. The elevated activities of DNA-dependent RNA-polymerase and reverse transcriptase were maintained within a long period of the animals life.
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PMID:[Mechanism of enzymatic imprinting induced in rats by an early postnatal administration of galactose]. 404 89


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