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)

Nucleoids isolated from Escherichia coli at low salt concentrations in the presence of spermidine (Kornberg et al., Proc. Natl. Acad. Sci. USA, 71, 3189-3193 (1974)) retain large amounts of protein and RNA and are, thus, potentially useful in structural and other studies. However, these preparations have neither been visualized nor extensively characterized with regard to their protein and other components. We have investigated this type of nucleoid preparation and here supply both light and electron microscope appearances and a description of the DNA-associated proteins. Light microscopy is used to follow the stages of nucleoid release and to demonstrate characteristically rounded nucleoids after chloramphenicol treatment of the cells from which the nucleoids were isolated. The nucleoids are "envelope-associated" particles. Electron microscopy shows an irregular central core that is partially covered with small, membranous vesicles. A significant fraction of the nucleoids have a characteristic doublet/dumbbell-shaped appearance by light microscopy. The nucleoids contain large amounts of protein and RNA in addition to DNA. The DNA and RNA are rendered acid-soluble by very low levels of nucleases, indicating an open structure. A small group of proteins, including H-NS, FIS, HU, and RNA polymerase, is released from the particles upon enzymatic digestion of the DNA.
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PMID:Isolation and characterization of spermidine nucleoids from Escherichia coli. 924 70

Nucleoids from Escherichia coli were isolated in the presence of spermidine at low salt concentrations. The nucleoids denature at relatively low temperatures or salt concentrations, yielding broad slowly sedimenting zones and/or macroscopic aggregates upon sucrose gradient centrifugation. Denaturation is accompanied by a loss of a characteristically compact shape as visualized by light and electron microscopy. Addition of polyethylene glycol or dextran prevents these changes, extending the range of stability of the isolated nucleoids to temperatures and ionic conditions like those which commonly occur in vivo. The effects of the polymers are consistent with stabilization by macromolecular crowding. Enzymatic digestion of the nucleoid DNA primarily releases three small proteins (H-NS, FIS, and HU) and RNA polymerase, as well as residual lysozyme from the cell lysis procedure. If isolated nucleoids are extracted with elevated salt concentrations under crowded, stabilized conditions, two of the proteins (HU and lysozyme) are efficiently removed and the compact form of the nucleoids is retained. These extracted nucleoids maintain their compact form upon reisolation into the initial uncrowded low-salt medium, indicating that HU, the most common "histone-like" protein of E. coli, is not a necessary component for maintaining compaction in these preparations.
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PMID:Stabilization of compact spermidine nucleoids from Escherichia coli under crowded conditions: implications for in vivo nucleoid structure. 924 71

rRNA transcription in Escherichia coli is activated by the FIS protein, which binds upstream of rrnp1 promoters and interacts directly with RNA polymerase. Analysis of the contribution of FIS to rrn transcription under changing physiological conditions is complicated by several factors: the wide variation in cellular FIS concentrations with growth conditions, the contributions of several other regulatory systems to rRNA synthesis, and the pleiotropy of fis mutations. In this report, we show by in vivo footprinting and Western blot analysis that occupancy of the rrnBp1 FIS sites correlates with cellular levels of FIS. We find, using two methods of measurement (pulse induction of a FIS-activated hybrid promoter and primer extension from an unstable transcript made from rrnBp1), that the extent of transcription activation by FIS parallels the degree of FIS site occupancy and therefore cellular FIS levels. FIS activates transcription throughout exponential growth at low culture density, but rrnp1 transcription increases independently of FIS immediately following upshift, before FIS accumulates. These results support the model that FIS is one of a set of overlapping signals that together contribute to transcription from rrnp1 promoters during steady-state growth.
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PMID:Activation of Escherichia coli rRNA transcription by FIS during a growth cycle. 951 22

Prokaryotic transcriptional activation often involves the formation of DNA microloops upstream of the polymerase binding site. There is substantial evidence that these microloops function to bring activator and polymerase into close spatial proximity. However additional functions are suggested by the ability of certain activators, of which FIS is the best characterised example, to facilitate polymerase binding, promoter opening and polymerase escape. We review here the evidence for the concept that the topology of the microloop formed by such activators is tightly coupled to the structural transitions in DNA mediated by RNA polymerase. In this process, which we term torsional transmission, a major function of the activator is to act as a local topological homeostat. We argue that the same mechanism may also be employed in site-specific DNA inversion.
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PMID:DNA microloops and microdomains: a general mechanism for transcription activation by torsional transmission. 964 81

The transcription factor FIS has been implicated in the regulation of several stable RNA promoters, including that for the major tRNALeu species in Escherichia coli, tRNA1Leu. However, no evidence for direct involvement of FIS in tRNA1Leu expression has been reported. We show here that FIS binds to a site upstream of the leuV promoter (centered at -71) and that it directly stimulates leuV transcription in vitro. A mutation in the FIS binding site reduces transcription from a leuV promoter in strains containing FIS but has no effect on transcription in strains lacking FIS, indicating that FIS contributes to leuV expression in vivo. We also find that RNA polymerase forms an unusual heparin-sensitive complex with the leuV promoter, having a downstream protection boundary of approximately -7, and that the first two nucleotides of the transcript, GTP and UTP, are required for formation of a heparin-stable complex that extends downstream of the transcription start site. These studies have implications for the regulation of leuV transcription.
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PMID:Activation of Escherichia coli leuV transcription by FIS. 1036 69

We recently identified Escherichia coli RNA polymerase (RNAP) mutants (RNAP beta' Delta215-220 and beta RH454) that form extremely unstable complexes with rRNA P1 (rrn P1) core promoters. The mutant RNAPs reduce transcription and alter growth rate-dependent regulation of rrn P1 core promoters, because the mutant RNAPs require higher concentrations of the initiating nucleoside triphosphate (NTP) for efficient transcription from these promoters than are present in vivo. Nevertheless, the mutants grow almost as well as wild-type cells, suggesting that rRNA synthesis is not greatly perturbed. We report here that the rrn transcription factor FIS activates the mutant RNAPs more strongly than wild-type RNAP, thereby compensating for the altered properties of the mutant RNAPs. FIS activates the mutant RNAPs, at least in part, by reducing the apparent K(ATP) for the initiating NTP. This and other results suggest that FIS affects a step in transcription initiation after closed-complex formation in addition to its stimulatory effect on initial RNAP binding. FIS and NTP levels increase with growth rate, suggesting that changing FIS concentrations, in conjunction with changing NTP concentrations, are responsible for growth rate-dependent regulation of rrn P1 transcription in the mutant strains. These results provide a dramatic demonstration of the interplay between regulatory mechanisms in rRNA transcription.
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PMID:Regulation of rRNA transcription is remarkably robust: FIS compensates for altered nucleoside triphosphate sensing by mutant RNA polymerases at Escherichia coli rrn P1 promoters. 1071 5

The wild-type Escherichia coli bgl promoter is silent in vivo but active in vitro. Silencing in vivo is directed by silencer sequences that flank the promoter, and requires nucleoid-associated protein H-NS and other unidentified cellular factors. Here we show that the DNA bending protein FIS is a repressor of the bgl promoter. Two FIS binding sites, centred at positions -52 and -27, overlap the CAP binding site and the -35 box respectively. FIS efficiently competes with CAP for binding to the wild-type promoter. However, FIS does not prevent binding of RNA polymerase. It interferes with the formation of a heparin-resistant complex and represses transcription initiation up to 40-fold. The presence of CAP has very little effect on the FIS-mediated repression of the wild-type bgl promoter in vitro. However, when a bgl promoter allele was tested that carries an improved CAP binding site (which leads to activation in vivo) CAP effectively counteracted repression by FIS in vitro. These results suggest that FIS contributes to silencing of the wild-type bgl promoter in vivo, presumably in the early exponential phase when FIS is predominantly expressed.
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PMID:Antagonistic control of the Escherichia coli bgl promoter by FIS and CAP in vitro. 1076 Jan 65

We have applied laser UV photo-footprinting to characterise kinetically complexes involving the activator protein FIS, RNA polymerase and the tyrT promoter of Escherichia coli. FIS photo-footprints strongly to three binding sites upstream of the core promoter. The polymerase photo-footprints in the near-consensus -35 hexamer on the non-template strand of DNA in a fashion similar to that of stable complexes involving the lacUV5 promoter. The kinetics of the interactions of polymerase alone with the tyrT promoter differ from those observed previously at the lacUV5 promoter. In the absence of FIS, we observe an upstream polymerase-induced signal at -122 within FIS site III that occurs subsequent to changes in the core promoter region and is strongly dependent on negative supercoiling. These observations support the proposal that the upstream region of the promoter is wrapped around the polymerase. We propose that the wrapped DNA allows the polymerase to overcome, at least in part, the barrier to DNA untwisting imparted by the G+C-rich discriminator. We further suggest that FIS plays a similar role and may facilitate polymerase escape.
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PMID:FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter. 1205 13

The stable RNA promoters of Escherichia coli are exquisitely sensitive to variations in the superhelical density of DNA. Previously, we have shown that binding of the DNA architectural protein FIS at the upstream activating sequences (UASs) of stable RNA promoters prevents the transcription complexes from inactivation induced by changes in the supercoiling level of DNA. Here, we identify a strong FIS binding site 89 bp upstream of the previously described cluster of FIS binding sites located between positions -64 and -150 in the rrnA P1 UAS. Binding of FIS to this 'far upstream sequence' allows the recruitment of additional FIS molecules to the region. We demonstrate that, upon DNA relaxation, the maintenance of promoter activity requires, in addition to UAS, the presence of the far upstream sequence. The far upstream sequence shows no effect in the absence of an intact cluster. This requirement for the integrity of the region encompassing the far upstream sequence and the UAS cluster is correlated with the in vitro modulation of binding of FIS to UAS and interaction of RNA polymerase with the UP element and the region around the transcriptional start point. Our results suggest that, at the rrnA P1 promoter, the entire region comprising the UAS and the far upstream sequence is involved in the assembly of the transcription initiation complex. We propose that the extensive engagement of upstream DNA in this nucleoprotein complex locally compensates for the lack of torsional strain in relaxed DNA, thus increasing the resistance of the promoter to global DNA relaxation.
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PMID:Buffering of stable RNA promoter activity against DNA relaxation requires a far upstream sequence. 1522 10

The synthesis of ribosomal RNAs in bacteria is tightly coupled to changes in the environment. This rapid adaptation is the result of several intertwined regulatory networks. The two proteins FIS and H-NS have previously been described to act as antagonistic transcription factors for rRNA synthesis. Here we provide evidence for another player, the regulatory protein LRP, which binds with high specificity to all seven Escherichia coli rRNA P1 promoter upstream regions (UAS). Comparison of the binding properties of LRP and H-NS, and characterization of the stabilities of the various complexes formed with the rRNA UAS regions revealed different binding modes. Binding studies with LRP and H-NS in combination demonstrated that the two proteins interacted with obvious synergism. The efficiency of LRP binding to the rRNA regulatory region is modified by the presence of the effector amino acid leucine, as has been shown for several other operons regulated by this transcription factor. The effect of LRP on the binding of RNA polymerase to the rrnB P1 promoter and in vitro transcription experiments indicated that LRP acts as a transcriptional repressor, thus resembling the activity of H-NS described previously. The results show for the first time that LRP binds to the regulatory region of bacterial rRNA promoters, and very likely contributes in combination with H-NS to the control of rRNA synthesis. From the known properties of LRP a mechanism can be inferred that couples rRNA synthesis to changes in nutritional quality.
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PMID:LRP and H-NS--cooperative partners for transcription regulation at Escherichia coli rRNA promoters. 1623 33


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