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 thrU(tufB) operon of Escherichia coli is endowed with a cis-acting region upstream of the promoter, designated UAS for Upstream Activator Sequence. A protein fraction has been isolated that binds specifically to DNA fragments of the UAS, thus forming three protein-DNA complexes corresponding to three binding sites on the UAS. It stimulates in vitro transcription of the operon by facilitating the binding of the RNA polymerase to the promoter. All three protein-DNA complexes contain one and the same protein. Dissociation constants for the three complexes have been determined, the lowest being in the sub-nanomolar range. The protein also binds to the UAS of the tyrT operon and to the UAS upstream of the P1 promoter of the rrnB operon, suggesting that transcription of the three operons, if not of more stable RNA operons, is activated by a common trans activator. We demonstrate that the E.coli protein FIS (Factor for Inversion Stimulation) also binds to the UAS of the thrU(tufB) operon forming three protein-DNA complexes. A burst of UAS- and FIS-dependent promoter activity is observed after reinitiation of growth of stationary cultures in fresh medium.
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PMID:The role of FIS in trans activation of stable RNA operons of E. coli. 169 Jan 24

The upstream activator sequence (UAS) of the thrU(tufB) operon, which is the target of the trans-activating protein FIS, has a bent structure. Here we show that the center of bending lies around position -95, between the two FIS-binding regions. Studies with fis+ and fis- cells show that FIS-induced bending of the UAS plays a major role in the trans-activation of the thrU(tufB) operon. This has been concluded from the finding that insertions of small DNA segments, comprising less than one or two complete helix turns, in the junction of the UAS and the RNA polymerase-binding site reduce transcription significantly. Partial restoration of transcriptional activity occurs when one or more full helix turns are inserted. These data are in line with but do not prove that a direct interaction between FIS and RNA polymerase is involved in trans-activation. A role of bending per se resulting from FIS/DNA interaction cannot be excluded.
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PMID:FIS-induced bending of a region upstream of the promoter activates transcription of the E coli thrU(tufB) operon. 176 17

The Escherichia coli DNA binding protein FIS activates stable RNA promoters during outgrowth of cells from stationary phase. The upstream activating sequences (UASs) of these promoters contain three highly conserved FIS binding sites positioned in helical register. Neither the apparent requirement for three sites nor the mechanism of FIS-mediated activation has been established. We demonstrate here that on saturation of its three binding sites in the UAS, FIS forms a specific nucleoprotein complex which 'traps' RNA polymerase (RNAP) at the promoter of the tyrT operon. This effect is abolished by a change in helical phasing between FIS sites II and III, which impaires cooperative interactions between DNA-bound FIS dimers. The sigma 70 subunit of RNAP stimulates the formation of higher order FIS complexes, a property that is indicative of protein-protein interactions. We propose that after initiation of transcription, the released sigma 70 subunit may be recaptured by the FIS nucleoprotein 'trap' and recycled in successive rounds of holoenzyme assembly. Such a mechanism could overcome transient limitations on the availability of sigma 70 or core polymerase after a prolonged stationary phase.
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PMID:FIS and RNA polymerase holoenzyme form a specific nucleoprotein complex at a stable RNA promoter. 753 15

The E. coli rrnB P1 promoter owes its strength, in part, to the transcriptional activator protein FIS. FIS binds to three sites upstream of the RNA polymerase (RNAP) binding site and increases transcription in vivo four to ten-fold. In this report, hydroxyl radical and DMS footprinting analyses show that FIS binds to its three sites along one side of the DNA helix, and that FIS bound at the promoter-proximal site (site I) and RNAP bound at the promoter are in close proximity. The binding of FIS at site I and RNAP at the promoter are mutually cooperative. These observations support a model for direct interaction between the FIS protein bound at site I and RNAP in transcription activation at rrnB P1. We also find that FIS does not bind cooperatively to its three sites upstream of rrnB P1, and that the relatively small activation associated with FIS bound at sites II and III does not result indirectly by facilitation of binding of FIS to site I.
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PMID:The transcriptional activator protein FIS: DNA interactions and cooperative interactions with RNA polymerase at the Escherichia coli rrnB P1 promoter. 784 12

DNA sequences upstream of the rrnB P1 core promoter (-10, -35 region) increase transcription more than 300-fold in vivo and in vitro. This stimulation results from a cis-acting DNA sequence, the UP element, which interacts directly with the alpha subunit of RNA polymerase, increasing transcription about 30-fold, and from a positively acting transcription factor, FIS, which increases expression another 10-fold. A DNA region exhibiting a high degree of intrinsic curvature has been observed upstream of the rrnB P1 core promoter and has thus been often cited as an example of the effect of bending on transcription. However, the precise position of the curvature has not been determined. We address here whether this bend is in fact related to activation of rRNA transcription. Electrophoretic analyses were used to localize the major bend in the rrnB P1 upstream region to position approximately -100 with respect to the transcription initiation site. Since most of the effect of upstream sequences on transcription results from DNA between the -35 hexamer and position -88, i.e. downstream of the bend center, these studies indicate that the curvature leading to the unusual electrophoretic behavior of the upstream region does not play a major role in activation of rRNA transcription. Minor deviations from normal electrophoretic behavior were associated with the region just upstream of the -35 hexamer and could conceivably influence interactions between the UP element and the alpha subunit of RNA polymerase.
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PMID:Localization of the intrinsically bent DNA region upstream of the E.coli rrnB P1 promoter. 803 62

We have extended our previous studies of the DNA sequences required for growth rate-dependent control of rRNA transcription in Escherichia coli. Utilizing a reporter system suitable for evaluation of promoters with low activities, we have found that the core promoter region of rrnB P1 (-41 to +1 with respect to the transcription initiation site) is sufficient for growth rate-dependent control of transcription, both in the presence and in the absence of guanosine 3'-diphosphate 5'-diphosphate (ppGpp). The core promoter contains the -10 and -35 hexamers for recognition by the sigma 70 subunit of RNA polymerase but lacks the upstream (UP) element, which increases transcription by interacting with the alpha subunit of RNA polymerase. It also lacks the binding sites for the positive transcription factor FIS. Thus, the UP element, FIS, and ppGpp are not needed for growth rate-dependent regulation of rRNA transcription. In addition, we find that several core promoter mutations, including -10 and -35 hexamer substitutions, severely reduce rrnB P1 activity without affecting growth rate-dependent control. Thus, a high activity is not a determinant of growth rate regulation of rRNA transcription.
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PMID:Growth rate-dependent control of the rrnB P1 core promoter in Escherichia coli. 807 Dec 40

The FIS protein is a transcription activator of rRNA and other genes in Escherichia coli. We have identified mutants of the FIS protein resulting in reduced rrnB P1 transcription activation that nevertheless retain the ability to bind DNA in vivo. The mutations map to amino acid 74, the N-terminal amino acid of the protein's helix-turn-helix DNA binding motif, and to amino acids 71 and 72 in the adjoining surface-exposed loop. In vitro analyses of one of the activation-defective mutants (with a G-to-S mutation at position 72) indicates that it binds to and bends rrnB P1 FIS site I DNA the same as wild-type FIS. These data suggest that amino acids in this region of FIS are required for transcription activation by contacting RNA polymerase directly, independent of any other role(s) this region may play in DNA binding or protein-induced bending.
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PMID:A positive control mutant of the transcription activator protein FIS. 875 36

The bacterial genome is present in the cell within a complex structure, the nucleoid. The nucleoid contains the genomic DNA, and molecules of RNA and proteins. The main proteins of the nucleoid are: RNA polymerase, topoisomerases and the histone-like proteins: HU, H-NS (H1), H, HLP1, IHF and FIS. The DNA molecule in the nucleoid is under helical tension or supercoiling and is organized into 43 +/- 10 topodomains. DNA supercoiling is generated by the activity of the topoisomerases and by DNA-protein interactions. In this review, we analize current knowledge in Escherichia coli about genome organization and proteins of the nucleoid.
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PMID:[The bacterial nucleoid]. 885 Mar 47

FIS, a site-specific DNA binding and bending protein, is a global regulator of gene expression in Escherichia coli. The ribosomal RNA promoter rrnB P1 is activated 3- to 7-fold in vivo by a FIS dimer that binds a DNA site immediately upstream of the DNA binding site for the C-terminal domain (CTD) of the alpha subunit of RNA polymerase (RNAP). In this report, we identify several FIS side chains important specifically for activation of transcription at rrnB P1. These side chains map to positions 68, 71 and 74, in and flanking a surface-exposed loop adjacent to the helix-turn-helix DNA binding motif of the protein. We also present evidence suggesting that FIS activates transcription at rrnB P1 by interacting with the RNAP alphaCTD. Our results suggest a model for FIS-mediated activation of transcription at rrnB P1 that involves interactions between FIS and the RNAP alphaCTD near the DNA surface. Although FIS and the transcription activator protein CAP have little structural similarity, they both bend DNA, use a similarly disposed activation loop and target the same region of the RNAP alphaCTD, suggesting that this is a common architecture at bacterial promoters.
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PMID:Molecular anatomy of a transcription activation patch: FIS-RNA polymerase interactions at the Escherichia coli rrnB P1 promoter. 900 76

FIS (factor for inversion stimulation) is a small dimeric DNA-bending protein which both stimulates DNA inversion and activates transcription at stable RNA promoters in Escherichia coli. Both these processes involve the initial formation of a complex nucleoprotein assembly followed by local DNA untwisting at a specific site. We have demonstrated previously that at the tyrT promoter three FIS dimers are required to form a nucleoprotein complex with RNA polymerase. We now show that this complex is structurally dynamic and that FIS, uniquely for a prokaryotic transcriptional activator, facilitates sequential steps in the initiation process, enabling efficient polymerase recruitment, untwisting of DNA at the transcription startpoint and finally the escape of polymerase from the promoter. Activation of all these steps requires that the three FIS dimers bind in helical register. We suggest that FIS acts by stabilizing a DNA microloop whose topology is coupled to the local topological transitions generated during the initiation of transcription.
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PMID:FIS activates sequential steps during transcription initiation at a stable RNA promoter. 921 6


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