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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 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

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

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

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

Transcription of the genes belonging to the phosphate (pho) regulon in Escherichia coli requires the specific activator protein PhoB, in addition to RNA polymerase containing the major sigma factor, sigma 70, which is encoded by rpoD. We previously isolated two mutant sigma 70s (D570G and E575K) that were specifically defective in transcribing the pho genes. The mutated sites were located near and within the first helix of the helix-turn-helix (HTH) motif or region 4.2 of sigma 70. To study further the role of the first helix of the HTH motif of sigma 70 in transcriptional activation by PhoB, we made a series of rpoD mutations that alter the motif and purified the mutant sigma 70 proteins. RNA polymerases containing the mutant sigma 70s Y571A, T572L, V576T, K578E and F580V showed reduced in vitro transcription from the pstS promoter, a representative pho promoter, in the presence of PhoB, whereas RNA polymerase containing another mutant sigma 70 (E574K) showed enhanced transcription from the promoter. Transcription from the activator-independent tac promoter and the pBR-P4 promoter, which is independent of PhoB and requires cAMP-CRP (cAMP receptor protein) for transcription, was affected at most only marginally by these sigma 70 mutations. These results provide further evidence that the first helix plays an important role in the specific interaction between RNA polymerase and PhoB protein bound to the pho promoters in transcriptional activation.
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PMID:Mutational analysis of the role of the first helix of region 4.2 of the sigma 70 subunit of Escherichia coli RNA polymerase in transcriptional activation by activator protein PhoB. 765 20

A series of gal promoter mutants has been used to compare the in vitro selectivities of the two forms of Escherichia coli RNA polymerase, E sigma 38 and E sigma 70. In the absence of the CRP-cAMP complex, E sigma 38 shows a strong preference for the ga/P1 promoter, whereas E sigma 70 preferentially initiates transcription from the ga/P2 promoter. E sigma 38 selectivity is not affected by the nature and position of the upstream sequences or by the phasing between synthetic upstream curved sequences and the -10 regions. In fact, all effects of mutations in the extended -10 region can be accounted for without evoking strong new sequence preferences for E sigma 38. Finally, both E sigma 38 and E sigma 70 initiate transcription from the ga/P1 promoter in the presence of CRP-cAMP complex and support direct cAMP-CRP activation at several CRP-dependent promoters.
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PMID:Selectivity of the Escherichia coli RNA polymerase E sigma 38 for overlapping promoters and ability to support CRP activation. 770 98

Sequence determinants responsible for promoter recognition by RNA polymerase holoenzyme containing sigma 38, the rpoS gene product, were analyzed. In a previous study [Tanaka et al. (1993) Proc. Natl. Acad. Sci. USA, 90, 3511-3515], Escherichia coli promoters were classified into three groups: promoters recognized only by RNA polymerase holoenzyme containing sigma 70 (E sigma 70); promoters recognized preferentially by that containing sigma 38 (E sigma 38); promoters recognized by both E sigma 70 and E sigma 38. As representatives of each group of promoter, we chose the alaS, fic and lacUV5 promoters. Making use of a restriction enzyme site inserted between the -10 and -35 hexamer sequences, promoters were divided into the upstream (UE) and downstream (DE) elements. These UEs and DEs were combined in all possible combinations and used for in vitro transcription reactions. Promoters containing DE from the fic or lacUV5 promoter were found to be recognized by E sigma 38, while those containing DE from the alaS promoter were not. Moreover, fic DE alone functioned as an efficient promoter for E sigma 38. Thus we conclude that the discrimination signal resides within the DE sequence. To test the activator response of E sigma 38, in vitro transcription reactions were also performed with the gal and lac promoters. For both CRP-responsive P1 promoters, E sigma 38 was found to be activated by the CRP-cAMP complex.
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PMID:Promoter determinants for Escherichia coli RNA polymerase holoenzyme containing sigma 38 (the rpoS gene product). 770 99

The lumP gene is linked to the lux operon, but runs in the opposite direction in Photobacterium leiognathi PL741. The gene order of the lumP and the lux operon is < -lumP-R & R-luxC-luxD-luxA-luxB-luxN-luxE- > (R & R: regulatory region). The nucleotide sequence of the regulatory region (827-bp) between the lumP and the lux operon was determined. Sequence analysis illustrates that the regulatory region includes two divergent promoter systems, PR-promoter system for the lux operon (R-operon) and PL-promoter system for the lumP or lum operon (L-operon). Functional analysis of the regulatory region shows that the PR- and PL-promoter systems both are able to lead the gene expression. The deletion experiment result elicits that the PR- and PL-promoter are coordinatively and negatively regulated; the PR- and PL-promoter might be competing for recognition by RNA polymerase to initiate transcription. The fact of the LumP responsible for the spectral blue shift in P. leiognathi implied that the lumP gene closedly linked to the lux operon is for coordinative regulation with the lux operon. In addition, the glucose repression on the PR-promoter system shows that the expression of the lux operon is regulated by cAMP-CRP induction in E. coli.
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PMID:Nucleotide sequence and functional analysis of regulatory region of the lumP and the lux operon from Photobacterium leiognathi. 776 66


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