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)

Short oligonucleotides corresponding to the 5'-terminal sequence of reovirus mRNAs were produced in vitro by virion-associated transcriptase activity. Both capped and uncapped oligonucleotides were synthesized in molar excess relative to mRNA. Yields of uncapped oligomers including ppG-C and ppG-C-U were severalfold greater than the homologous capped structures. In partial reaction mixtures that were nonsupportive for mRNA chain elongation, capped oligomer synthesis was increased. Similarly, oligonucleotide formation was differentially resistant in viral preparations that were inactivated with respect to mRNA synthesis by modification of the genome RNA by dimethyl sulfate alkylation or psoralen photoreaction. The results suggest that reovirus mRNA synthesis involves excessive initiation by reiterative transcription of promoter sites by the reovirus polymerase. Only a small fraction of the resulting oligonucleotides are capped and extended to form full length mRNAs during a subsequent elongation step which is apparently mediated by transcriptase molecules that escape the reiterative phase of transcription.
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PMID:Excess synthesis of viral mRNA 5-terminal oligonucleotides by reovirus transcriptase. 724 Feb 22

Footprinting studies with the purine-modifying reagent dimethyl sulfate and with the single-stranded DNA probing reagent potassium permanganate were carried out in isolated mitochondria from rat liver. Dimethyl sulfate footprinting allowed the detection of protein-DNA interactions within the rat analogues of the human binding sites for the transcription termination factor mTERF and for the transcription activating factor mt-TFA. Although mTERF contacts were localized only at the boundary between the 16S rRNA/tRNA(Leu)UUR genes, multiple mtTFA contacts were detected. Contact sites were located in the light and the heavy strand promoters and, in agreement with in vitro footprinting data on human mitochondria, between the conserved sequence blocks (CSB) 1 and 2 and inside CSB-1. Potassium permanganate footprinting allowed detection of a 25-base pair region entirely contained in CSB-1 in which both strands were permanganate-reactive. No permanganate reactivity was associated with the other regions of the D-loop, including CSB-2 and -3, and with the mTERF contact site. We hypothesize that the single-stranded DNA at CSB-1 may be due to a profound helix distortion induced by mtTFA binding or be associated with a RNA polymerase pause site. In any case the location in CSB-1 of the 3' end of the most abundant replication primer and of the 5' end of the prominent D-loop DNA suggests that protein-induced DNA conformational changes play an important role in directing the transition from transcription to replication in mammalian mitochondria.
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PMID:Identification by in Organello footprinting of protein contact sites and of single-stranded DNA sequences in the regulatory region of rat mitochondrial DNA. Protein binding sites and single-stranded DNA regions in isolated rat liver mitochondria. 755 32

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

In earlier work, we demonstrated that 5'-CG-3' methylation inhibits the transcriptional activity of human Alu elements associated with the alpha 1-globin and the angiogenin genes in a cell-free transcription system from HeLa nuclear extracts. These studies have been extended to different Alu sequences and to investigations on the mechanism involved in transcriptional silencing by methylation. By comparing the results of DNase I and dimethyl sulfate (DMS) in vitro footprinting on a consensus sequence in the RNA polymerase III promoter control B region between the unmethylated and the 5'-CG-3' methylated B box, evidence has been adduced for effects of 5'-CG-3' methylation on the interaction of specific nuclear proteins with DNA sequences in the B control region of the Alu elements. These results are consistent with the interpretation that the 5'-CG-3' methylation interferes with the binding of proteins that are essential for the function of the B control region in these RNA polymerase III-transcribed elements, and that promoter methylation thus inhibits transcription.
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PMID:Transcriptional silencing of human Alu sequences and inhibition of protein binding in the box B regulatory elements by 5'-CG-3' methylation. 787 14

Down regulation of the c-myc gene is a prerequisite for the differentiation of a number of cell types. Studies have shown that two mechanisms of inactivation are involved in c-myc repression: a block of the elongation of RNA polymerase followed by transcriptional inactivation mediated through promoter sequences. In this study DMS in vivo footprinting was performed on the P2 promoter region of c-myc in differentiated and undifferentiated HL60 cells. A differentiation-specific footprint was observed at G residues immediately upstream of the TATA box. This observation occurred only in cells differentiated for 48 hours or more and hence is likely to be involved in the repression of c-myc by promoter inactivation.
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PMID:Changes in in vivo protein-DNA interactions occur at the c-myc P2 promoter during differentiation. 828 Jan 53

Activation of the spoIIG promoter at the onset of sporulation in Bacillus subtilis requires the regulatory protein, Spo0A, which binds to two sites in the promoter, sites 1 and 2. Phosphorylation of Spo0A is essential for the initiation of sporulation. Therefore, we examined the role of Spo0A phosphorylation in spoIIG promoter activation. Phosphorylation of Spo0A stimulated transcription from the spoIIG promoter in vitro. In DNAse I footprinting experiments with the spoIIG promoter, we found that phosphorylation of Spo0A increased its affinity for site 2 more than for site 1, which is the site to which nonphosphorylated Spo0A binds most avidly. This result could not be explained by increased cooperativity between Spo0A bound at sites 1 and 2 because the increased affinity for site 2 by phosphorylated Spo0A was also observed with a deletion derivative of the spoIIG promoter containing only site 2. We have located Spo0A-binding sequences in the spoIIG promoter by DMS protection assays and mutational analysis, and found that site 1 contains one higher-affinity binding sequence whereas site 2 contains two weaker-binding sites. Two substitutions in site 2 of the spoIIG promoter that change the sequence to be more like an optimal Spo0A-binding site were found to increase promoter activity. Moreover, phosphorylation of Spo0A was not required in vivo for activation of the spoIIG promoter containing these strong binding sites. The results suggest that the primary role for phosphorylation of Spo0A is to increase its affinity for specific sites rather than to activate an activity of Spo0A that acts on RNA polymerase at promoters.
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PMID:Phosphorylation of Bacillus subtilis transcription factor Spo0A stimulates transcription from the spoIIG promoter by enhancing binding to weak 0A boxes. 828 22

Regulation of transcriptional initiation of the Tn21 mercury resistance (mer) operon occurs at the divergent promoter region lying between the structural genes (merTPCAD) and a regulatory gene (merR). During repression, both promoters are negatively regulated by MerR bound to a dyadic operator located between the -10 and -35 hexamers of PTPCAD. Upon Hg(II) induction, MerR activates transcription only from PTPCAD and continues to repress transcription from PR. Using in vivo dimethyl sulfate and KMnO4 footprinting of the merOP region of strains carrying wild-type MerR or MerR mutants, we have dissected the steps in MerR-mediated positive and negative regulation of the divergent mer promoters. The greater sensitivity of primer extension footprinting allowed the resolution of details previously undetectable in vivo. Two MerR mutants unable to bind merOP DNA allow RNA polymerase to form an open complex preferentially at PR. The intensity of the PR open complex is considerably less than that which occurs upon MerR-Hg(II) activation of PTPCAD and considerably greater than that which occurs at PTPCAD when MerR is deleted; this is the first in vivo estimate of the relative strengths of these two promoters. Although retaining the wild-type capacity to sequester RNA polymerase in the closed complex, the four MerR mutants defective in Hg(II) binding do not distort the dyad DNA or foster open complex formation when the inducer is added. Two activation-defective MerR mutants foster closed complex formation as well as wild-type, but they do not distort the dyad DNA or foster open complex formation when the inducer is added, although they are also able to bind Hg(II). Two semiconstitutive inducible MerR mutants differ from each other in that one (which lies nearer the COOH terminus) does distort the dyad center upon Hg(II) induction, whereas the other (which lies in near the center of merR) exhibits no dyad distortion upon induction. Paradoxically, despite their relatively strong semiconstitutive expression of mer-lac transcriptional fusions, neither of these mutants has a detectable open complex in the absence of added Hg(II).
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PMID:In vivo DNA-protein interactions at the divergent mercury resistance (mer) promoters. I. Metalloregulatory protein MerR mutants. 842 39

Transcription of the Tn21 mercury resistance (mer) operon is regulated by MerR which represses and activates the mer structural genes (merTPCAD) in the absence and presence of Hg(II), respectively. The promoter for the structural genes (PTPCAD) is divergently overlapped with the promoter for the regulatory gene (PR), and a dyadic operator lies between the -10 and -35 hexamers of PTPCAD. Using in vivo dimethyl sulfate and KMnO4 footprinting of mutant mer operator-promoter (merOP) DNA to observe MerR and RNA polymerase-mediated interactions with the merOP region, we have identified three distinct domains within the palindromic mer operator. Dyad domain I consists of the outermost bases on the left arm of the operator palindrome whose alteration causes a shift, but apparently not a major loss, in occupancy by MerR, and no decrease in RNA polymerase occupancy. Mutants in dyad domain I are semiconstitutive but support additional Hg(II)-induced open complex formation at PTPCAD. Dyad domain II consists of the four highly conserved inner bases ( ... GTAC ... GTAC ... ) of the seven-base interrupted dyad, alteration of which severely modifies both MerR and RNA polymerase contacts in the promoter region. Mutants in domain II generally allow constitutive open complex formation at PR. One unusual mutant of this group retains most of the wild-type dyad's ability to repress both promoters but is unable to support activation at PTPCAD in response to Hg(II), indicating that MerR undergoes a conformational change and that the required base contacts for activation are different than those for repression. Dyad domain III is tentatively defined by a mutant in the outermost base of the right palindrome arm which is unaffected in either MerR or RNA polymerase occupancy, however, a second lesion within the PTPCAD -10 hexamer of this mutant limits effective open complex formation. Other mutations lying solely within the -10 RNA polymerase recognition hexamer of PTPCAD are similarly competent in both MerR and RNA polymerase binding, but inadequate for open complex formation. One such mutant also affects the overlapping -10 hexamer of PR and results in reduced occupancy by both MerR and RNA polymerase, likely as a result of inefficient transcriptional initiation of merR mRNA. Finally, mutations affecting the -35 hexamer of PTPCAD bind MerR but not RNA polymerase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:In vivo DNA-protein interactions at the divergent mercury resistance (mer) promoters. II. Repressor/activator (MerR)-RNA polymerase interaction with merOP mutants. 842 40

Transcription from the middle promoter, Pm, of phage Mu is initiated by Escherichia coli RNA polymerase holoenzyme (E sigma 70; RNAP) and the phage-encoded activator, Mor. Point mutations in the spacer region between the -10 hexamer and the Mor binding site result in changes of promoter activity in vivo. These mutations are located at the junction between a rigid T-tract and adjacent, potentially deformable G + C-rich DNA segment, suggesting that deformation of the spacer region may play a role in the transcriptional activation of Pm. This prediction was tested by using dimethyl sulfate and potassium permanganate footprinting analyses. Helical distortion involving strand separation was detected at positions -32 to -34, close to the predicted interface between Mor and RNAP. Promoter mutants in which this distortion was not detected exhibited a lack of melting in the -12 to -1 region and reduced promoter activity in vivo. We propose that complexes containing the distortion represent stressed intermediates rather than stable open complexes and thus can be envisaged as a transition state in the kinetic pathway of Pm activation in which stored torsional energy could be used to facilitate melting around the transcription start point.
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PMID:Distortion in the spacer region of Pm during activation of middle transcription of phage Mu. 879 Mar 43

The Bradyrhizobium japonicum fixRnifA operon is transcribed from two promoters: fixRp1, a -24/-12 promoter recognized by the sigma54-holoenzyme form of the RNA polymerase, and fixRp2, a -35/-10 promoter that is transcribed by a second, unidentified, form of RNA polymerase holoenzyme. The fixRp1 promoter is autoregulated during microaerobiosis by NifA, whereas fixRp2 is also activated, but by a different regulatory protein. The main transcription start sites for these promoters are just two nucleotides apart, such that the conserved -12 and -10 regions of fixRp1 and fixRp2, respectively, must overlap each other, whereas the -24 and -35 regions lie one DNA helical turn apart. Using in vivo genomic dimethyl sulfate and KMnO4 footprinting, we showed that the promoter region is differentially protected, depending upon which holoenzyme is bound. Mutagenesis analyses indicated that positions from -12 to -14 are critical for the activity of both promoters, whereas mutations at -10 and -11 affected mainly fixRp2 expression. When the sequence of the putative -35 region of fixRp2 was modified to match the putative consensus, expression from this promoter was increased 3-fold and the reactivity toward KMnO4, but not the transcriptional start site, moved two nucleotides further upstream, indicating that the altered promoter forms a different open complex. Additionally, we detected NifA-dependent methylation protection of two atypical NifA binding sites and protection of guanine -75. The latter residue is located in a region critical for fixRp2 promoter activation. The results present direct physical evidence of the complexity of the organization, regulation, and function of the fixRnifA promoter region.
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PMID:In vivo genomic footprinting analysis reveals that the complex Bradyrhizobium japonicum fixRnifA promoter region is differently occupied by two distinct RNA polymerase holoenzymes. 944 77


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