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)

In a wide variety of nitrogen-fixing organisms among the Purple Bacteria (large division of Gram-negative bacteria) the nitrogen fixation (nif) operons are transcribed by an alternative holoenzyme form of RNA polymerase, sigma 54-holoenzyme. Transcription depends on the activator protein NIFA (nitrogen fixation protein A), which catalyzes isomerization of closed complexes between this polymerase and a promoter to transcriptionally productive open complexes. NIFA-mediated activation of transcription from the nifH promoter of Klebsiella pneumoniae is greatly stimulated by the integration host factor IHF, which binds to a site between the upstream binding site for NIFA and the promoter, and bends the DNA. IHF fails to stimulate activation of transcription from this promoter by another activator of sigma 54-holoenzyme, NTRC (nitrogen regulatory protein C), which lacks a specific binding site in the nifH promoter region. As predicted, if the IHF-induced bend facilitates interaction between NIFA and sigma 54-holoenzyme, substitution of an NTRC-binding site for the NIFA-binding site allowed IHF to stimulate NTRC-mediated activation of transcription from the nifH promoter. The stimulation was of the same order of magnitude as that for NIFA in the native configuration of the promoter-regulatory region (up to 20-fold). With purified NTRC and the substitution construct we could demonstrate that stimulation by IHF in a purified transcription system was comparable to that in a crude coupled transcription-translation system, indicating that the stimulation in the crude system could be accounted for by IHF. The IHF stimulation was observed on linear as well as supercoiled templates, indicating that the geometric requirements are relatively simple. We have attempted to visualize the arrangement of proteins on DNA fragments carrying the nifH promoter-regulatory region of K. pneumoniae by electron microscopy. IHF stimulated NIFA-mediated activation of transcription from the nifH and nifD promoters of Bradyrhizobium japonicum and less so from the nifH promoters of Rhizobium meliloti and Thiobacillus ferrooxidans, consistent with previous observations that stimulation is greatest at promoters that are weak binding sites for sigma 54-holoenzyme in closed complexes.
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PMID:Role of integration host factor in stimulating transcription from the sigma 54-dependent nifH promoter. 140 79

Transcription of the Escherichia coli glnHPQ operon, which encodes components of the high-affinity glutamine transport system, is activated by nitrogen regulator I (NRI)-phosphate in response to nitrogen limitation. NRI-phosphate binds to sites upstream from the sigma 54-dependent glnHp2 promoter and activates transcription by catalyzing the isomerization of the closed sigma 54-RNA polymerase promoter complex to an open complex. On linear DNA, the initiation of glnHp2 transcription requires in addition to NRI-phosphate the presence of integration host factor (IHF), which binds to a site located between the NRI-binding sites and the promoter. On supercoiled DNA, IHF does not play an essential role, but enhances the activation of transcription by NRI-phosphate. We found that at a mutant glnHp2 promoter with increased affinity for sigma 54-RNA polymerase, the initiation of transcription can be activated equally well by NRI-phosphate in the presence or absence of IHF. Binding of IHF to its site does not increase the binding of sigma 54-RNA polymerase to the glnHp2 promoter; instead, our data suggest that IHF bends the DNA to align the activator with the closed sigma 54-RNA polymerase promoter complex to facilitate the interactions that result in open complex formation. In the absence of IHF, NRI-phosphate can activate transcription whether its binding sites are on the same face of the DNA helix as the sigma 54-RNA polymerase or on the opposite face. IHF enhances transcription when the three proteins are located on the same face of the helix, but strongly inhibits transcription when any one of the proteins is located on the opposite face.
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PMID:Positive and negative effects of DNA bending on activation of transcription from a distant site. 143 5

We have identified two intrinsically bent regions of DNA which flank the transcription start site of the rRNA gene from Physarum polycephalum. DNA fragments from both regions were analyzed by circular permutation polyacrylamide gel electrophoresis assay and computer modeling. Both types of analysis indicate that one fragment contains a relatively simple bend centered about 160 base pairs (bp) upstream of the transcription start site while the other fragment contains multiple bends, the most prominent of which is centered about 150 bp downstream of the start site. According to both gel mobilities and computer modeling we estimate that the net bending in each is about 45 degrees. These fragments were studied in detail by varying parameters of electrophoresis that are known to affect bending. Previous work indicates that anomalous mobility should decrease when temperature or ethidium bromide concentration is increased, whereas anomalous mobility should increase when polyacrylamide gel percentage is increased. The anomalous mobility of both fragments decreases as temperature is raised from 4 to 65 degrees C, although the bent structure centered at -160 bp is more temperature labile than the bend at +150 bp. Strikingly different behavior was observed for the two fragments as the polyacrylamide concentrations was varied. As polyacrylamide concentrations are increased from 6 to 10%, the anomalous mobility of the bend centered at -160 bp increases while that of the bend centered at +150 bp decreases. The bend centered at +150 bp is "straightened" at all ethidium concentrations tested. In sharp contrast and unexpectedly, the anomalous migration of the bend centered at -160 bp increases dramatically in 0.1 micrograms/ml ethidium bromide. Many of the mobility differences we observe suggest that the two regions studied represent structurally distinct forms of bent DNA. The location of these strongly bent regions on either side of a RNA polymerase I transcription start site suggests important roles for such structures in chromatin structure and transcription initiation.
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PMID:Intrinsically bent DNA flanks both sides of an RNA polymerase I transcription start site. Both regions display novel electrophoretic mobility. 157 26

We have shown that a transcriptional repressor protein can regulate promoter activity via DNA bending by using the pLS1 plasmid promoter PII (which has intrinsic curvature upstream of its -35 box) and the plasmid-encoded repressor protein RepA (which strongly bends DNA). Substitution of the curved region for a straight DNA fragment containing the RepA target resulted in increased (or decreased) gene expression when RepA was supplied in trans: enhanced gene expression was evident when the target of RepA and the promoter were on the same face of the DNA helix; repression was found when they were on opposite faces of the DNA. In vitro activation of transcription from PII was observed when supercoiled DNA was used as template, but not with linear molecules. We propose that promoter activity can be regulated by the proper positioning (in or out of phase) of an induced DNA bend with the RNA polymerase recognition sites.
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PMID:The RepA repressor can act as a transcriptional activator by inducing DNA bends. 202 40

A limited number of deletion/insertions and a point mutation in the -35 region of the p'R promoter of phage lambda were examined and found to influence both transcription and its repression by the integration host factor (IHF). Positive effects on transcription (in the absence of IHF) are small (up to 1.4-fold) and are caused by a deletion-substitution upstream of the -35/ihf site. Up to three base changes in the -35 promoter element seem to be tolerated, with only a small negative effect on transcription. In some cases, effective transcription requires supercoiling of such mutant template. Since an ihf sequence overlaps the -35 region of p'R, IHF represses transcription. Repression is correlated with IHF binding and consequent DNA bending, as assessed by gel retardation experiments. Nine p'R mutants were tested for their IHF binding and repression; the results confirm the consensus sequence, 5'-W2WWWWN7WWWWCARNWN2TTR derived from the hydroxyl radical footprinting, where the bold letters indicate the IHF-protected bases and W is A or T, R is A or G and N represents A, T, G or C. Perhaps surprisingly, some mutations just upstream or downstream of this ihf sequence also affect IHF binding, as observed not only for the pR'/ihf but also for the att H' site of lambda. Supercoiling in some cases permits the IHF-mediated repression to be overcome, probably by increasing the RNA polymerase binding and/or decreasing the interaction with IHF. All our data are consistent with a model which assumes that IHF initially binds to one or two ihf contact points depending on preexisting DNA topology, bends DNA, and completes the remaining contacts while finally adjusting the DNA conformation to establish the best fit within the minor groove of the double helix. Effective IHF repression of transcription would thus depend on several factors, including: (1) the sequence, and (2) the initial conformation of the ihf site, together with (3) the capacity of IHF to compete with RNA polymerase for the overlapping binding sites.
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PMID:Alterations in the p'R promoter of coliphage lambda modify both its activity and interaction with the integration host factor (IHF). 214 67

The regulatory protein NIFA activates transcription of nitrogen fixation (nif) operons by the sigma 54 holoenzyme form of RNA polymerase. NIFA from Klebsiella pneumoniae activates transcription from the nifH promoter in vitro; in addition, the integration host factor, IHF, binds between the nifH promoter and an upstream binding site for NIFA. We demonstrate here that IHF greatly stimulates NIFA-mediated activation of nifH transcription in vitro and thus that the two factors are functionally synergistic. Electron micrographs indicate that IHF bends the DNA in the nifH promoter regulatory region. Although IHF binds close to the nifH promoter, it does not directly stimulate binding of sigma 54 holoenzyme. Rather, the IHF-induced bend may facilitate productive contacts between NIFA and sigma 54 holoenzyme that lead to the formation of open complexes. IHF binds to nif promoter regulatory regions from a variety of organisms within the phylum "purple bacteria," suggesting a general ability to stimulate NIFA-mediated activation of nif transcription.
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PMID:The integration host factor stimulates interaction of RNA polymerase with NIFA, the transcriptional activator for nitrogen fixation operons. 220 75

Transcription factor IIIA (TFIIIA), the canonical zinc-finger protein, is a protein of relative molecular mass 39,000 (39K) that is required for transcription of 5S-ribosomal subunit genes in Xenopus. It binds in a sequence-specific manner to the internal control region of the 5S gene (see Fig. 1) and facilitates transcription of the gene by RNA polymerase III. It also binds to the 5S gene product to form a 7S ribonucleoprotein particle. In oocytes the 7S particle acts as a storage form of the RNA to be utilized later in development. TFIIIA binds to DNA through its 30 K N-terminal domain, which contains nine zinc-fingers. TFIIIA was the first protein described to have this type of DNA binding motif, but numerous other proteins have now been shown to have zinc-finger domains. A structure for a single zinc-finger from the yeast protein ADR1, was recently proposed based on two-dimensional NMR data (ref. 8), and a similar structure was proposed based on comparison with crystal structures of other metalloproteins. Although models for the interaction of TFIIIA with the 5S-ribosomal gene DNA have been proposed, based on nuclease digestion and methylation interference data, little precise structural information is available for TFIIIA and the physical basis for the interaction of zinc-fingers with DNA is not understood. Using both circular permutation and circularization assays we provide convincing biochemical evidence that TFIIIA bends the DNA at the internal promoter of the 5S gene.
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PMID:Transcription factor IIIA induced bending of the Xenopus somatic 5S gene promoter. 275 11

A Saccharomyces cerevisiae RNA polymerase III transcription factor was previously shown to bind stably to tRNA genes. This transcription factor has been further purified on the basis of its large size and its binding to a S. cerevisiae tRNALeu3 gene has been examined by electron microscopy. Site-specific binding of the factor to the tRNALeu3 gene sharply bends the DNA.
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PMID:Electron-microscopic examination of the binding of a large RNA polymerase III transcription factor to a tRNA gene. 390 64

Integration host factor (IHF) of Escherichia coli is an asymmetric histone-like protein that binds and bends the DNA at specific sequences. IHF functions as an accessory factor in a wide variety of processes including replication, site-specific recombination and transcription. In many of these processes IHF was shown to act as an architectural element which helps the formation of nucleo-protein complexes by bending of the DNA at specific sites. This MicroReview shows how such a structural role of IHF can influence the initiation of transcription. In addition, it summarizes the evidence indicating that IHF can stimulate transcription via a direct interaction with RNA polymerase and explores the possibility that the asymmetry of the IHF protein might reflect such an interaction.
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PMID:The regulation of transcription initiation by integration host factor. 765 Nov 28

Recent discoveries of activator proteins that distort DNA but bear no obvious activation domains have focused attention on the role of DNA structure in transcriptional regulation. Here we describe how the transcription factor MerR can mediate repression as well as activation through stereospecific modulation of DNA structure. The repressor form of MerR binds between the -10 and -35 promoter elements of the bacterial mercury-detoxification genes, PT, allowing RNA polymerase to form an inactive complex with PT and MerR at this stress-inducible promoter. Upon mercuric ion binding, Hg-MerR converts this polymerase complex into the transcriptionally active or 'open' form. We show here that MerR bends DNA towards itself in a manner similar to the bacterial catabolite-activator protein CAP, namely at two loci demarked by DNase I sensitivity, and that the activator conformation, Hg-MeR, relaxes these bends. This activator-induced unbending, when coupled with the previously described untwisting of the operator, remodels the promoter and makes it a better template for the poised polymerase.
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PMID:DNA-bend modulation in a repressor-to-activator switching mechanism. 788 78


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