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 Saccharomyces cerevisiae transcription factors (TF) IIIB and IIIC assemble onto their respective DNA-binding sites on the SUP4 tRNA(Tyr) gene at 0 degrees C. RNA polymerase III specifically associates at 0 degrees C with this TFIIIC-TFIIIB-DNA complex to form a stable "closed" promoter complex in which the DNA surrounding the transcriptional start retains its duplex form. Promoter "opening" is a temperature-dependent and readily reversible process that involves up to 22 unwound base-pairs of DNA, and can be followed by analyzing the hyperreactivity of thymine to KMnO4 oxidation. This promoter opening increases progressively from 10 degrees C to 40 degrees C, with at least two regions within the transcription bubble appearing to melt independently. In contrast, the temperature dependence of forming an initiated transcription complex containing a 17 nucleotide nascent RNA chain displays a sharp transition between 10 degrees C and 15 degrees C. When RNA polymerase initiates transcription under conditions that limit the nascent RNA chain to less than six nucleotides, there is no displacement of the transcription bubble. These transcription complexes are distinguishable from "open" promoter complexes in their maintenance of the transcription bubble at 0 degrees C, and from transcription complexes with more extended (17 nucleotide) RNA chains in their sensitivity to disruption by heparin. In light of recent results by others that demonstrate a requirement for an RNA transcription factor in a Bombyx mori-based in vitro RNA polymerase III transcription system, we have searched for a comparable component in the S. cerevisiae-derived system. We show that if an RNA component is required in the yeast-derived system, it is not susceptible to inactivation by massive amounts of micrococcal nuclease, RNase A, or RNase T1.
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PMID:Formation of open and elongating transcription complexes by RNA polymerase III. 161 62

During transcription, positive DNA supercoils generated ahead of RNA polymerase could theoretically uncoil the negative DNA supercoils associated with nucleosomes and thereby decondense the chromatin fiber in preparation for RNA polymerase passage. Here we examine the effect of positive DNA supercoiling on the structure of yeast 2-microns minichromosomes. We utilized a conditional topoisomerase mutant expressing Escherichia coli topoisomerase I to convert the DNA supercoiling state from negative to positive in vivo. Minichromosomes containing positively supercoiled DNA exhibited a striking increase in DNase I sensitivity. They also displayed additional micrococcal nuclease cleavage sites but yielded nearly typical nucleosomal ladders after extensive digestion. Upon in vitro relaxation with eukaryotic topoisomerase I, the minichromosomes remained DNase I sensitive but were converted to negative DNA supercoiling with a slightly increased linking number compared to typical minichromosomes, thus indicating the presence of bound histones. Therefore, positive DNA supercoiling provides a mechanism for generating, but is not required for maintaining, a conformation in chromatin characteristic of highly transcribed genes.
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PMID:Positive DNA supercoiling generates a chromatin conformation characteristic of highly active genes. 194 86

In vitro studies have demonstrated that the estrogen receptor (ER) can bind to the rat PRL estrogen response element (ERE) located 1700 basepairs upstream of the transcriptional start site. However, the mechanism by which the receptor-DNA complex influences the activity of RNA polymerase located in the promoter region is not understood. To begin investigating this process, we developed cell lines derived from GH3 cells that contain steroid-responsive bovine papillomavirus minichromosomes. Within these minichromosomes is a hybrid gene composed of the 5' flanking region of the PRL gene, driving the expression of the Tn5 gene. The episomal PRL DNA sequences responded to 17 beta-estradiol (E2) by increasing the rate of Tn5 gene transcription. Nucleosome mapping experiments using micrococcal nuclease demonstrated that nucleosome-like structures were assembled on the minichromosome in an ordered array separated by 150-200 basepairs of DNA. Novel S1 nuclease as well as DNase-I-hypersensitive sites in the chromatin of the promoter and distal regulatory regions of the episomal PRL gene were detected by indirect end-labeling studies. The nuclease hypersensitive sites in the distal region containing the ERE were modified after treatment of the cells with either E2 or the antiestrogen 4-hydroxytamoxifen. However, only E2 treatment of cells resulted in an increase in the nuclease hypersensitivity of the promoter region and induced gene expression, while antiestrogen treatment had no effect on either parameter. This suggests that complex interactions between factors located at the distal and proximal regulatory regions ultimately determine the transcriptional response of the PRL gene to E2.
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PMID:An interaction between the 5' flanking distal and proximal regulatory domains of the rat prolactin gene is required for transcriptional activation by estrogens. 196 74

The developmentally regulated sea urchin early histone gene repeat (SUEHGR) from Strongylocentrotus purpuratus was isolated as chromatin by nucleoprotein hybridization. This technique is a novel method to isolate specific sequences as chromatin. Because the purification scheme is based only on the gene sequence and is independent of other physical properties such as protein composition and transcriptional activity, we were able to isolate the same gene in different functional states. Gene size chromatin fragments were solubilized by restriction endonuclease digestion of cell nuclei. Using T7 gene 6 exonuclease, the 3'termini of the fragments were exposed and then hybridized in solution to a biotinylated oligonucleotide complementary to one end of the SUEHGR fragment. The hybrids were bound to an Avidin D matrix. DTT cleavage of the biotin linker yielded a chromatin fraction greater than 700 fold enriched in SUEHGR. Overall yields were between 2% and 15%. The purity of the isolated material was independently measured to be greater than 80%. The homogeneous native structure of the inactive genes was preserved as shown by electron microscopy and micrococcal nuclease digestion of the purified SUEHGR. Minor heterogeneity was observed for the purified active genes by micrococcal nuclease digestion but the main features of the active chromatin were preserved during isolation. This isolation offers the first opportunity to study the structure of an RNA polymerase II gene at different stages of the cell cycle and development.
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PMID:Nucleoprotein hybridization: a method for isolating active and inactive genes as chromatin. 203 Sep 47

During transcription, positive and negative superhelical stresses are generated on a DNA template which could potentially affect nucleosomal structure. When transcription was performed on a closed circular plasmid containing nucleosomes, using T7 RNA polymerase and topoisomerase I, nucleosomal structure was lost from the DNA. Nucleosome content was assayed by analyzing both the topological state of the DNA and the nuclease-resistant fragments produced by micrococcal nuclease and DNase I treatment. This nucleosome dissolution required positive superhelical stress as evidenced by the requirement that the extended RNA transcript remain associated with the polymerase during the transcription process. Rates of transcription were found to be independent of whether the nucleosomes dissolved. When transcription was performed in the absence of topoisomerase I, nucleosome reformation occurred very rapidly. This observation suggests that negative superhelical stress, induced in the wake of polymerase action, facilitates nucleosome reformation.
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PMID:In vitro evidence that transcription-induced stress causes nucleosome dissolution and regeneration. 217 Mar 57

RNA synthesis in the nuclei of liver from newly hatched (4-d-old) chicks is enhanced by intake of food. The enhanced synthesis was ascribed not to an increase in the activity of solubilized DNA-dependent RNA polymerase but to an increase in the initiation of RNA synthesis. Enhanced RNA synthesis in fed chicks was accompanied by greater susceptibility of nuclei to digestion by micrococcal nuclease. Salt extraction abolished the difference in nuclease sensitivity between the fed and fasted groups. Reconstitution with either 0.35 M NaCl extracts or high mobility group (HMG) nonhistone proteins restored digestion susceptibility, but changing the source of extracted proteins did not equalize the extent of digestion in nuclei from livers of fed and fasted chicks. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of HMG proteins revealed the presence of HMGs 1 and 2 as well as a 38,000-dalton protein. The nuclear HMG protein content in fed chicks was greater than that of fasted chicks (121 +/- 17 micrograms/mg DNA vs. 31 +/- 12 micrograms/mg DNA). The electron microscopic examination of hepatocyte nuclei revealed the enlargment of nucleoli and scarcity of aggregated heterochromatin structures in the fed chicks as compared with the fasted chicks. These morphological features are compatible with the high transcriptional activity in liver of fed chicks.
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PMID:Enhancement of RNA synthesis in chick liver by food intake: possible role of high mobility group nonhistone proteins. 241 21

Infection of cultured cells of Drosophila melanogaster with black beetle virus (BBV) induces an RNA polymerase that is bound to cellular particulate material in a complex with a template RNA. We have solubilized the polymerase by treatment of the relevant particulates with detergents such as dodecyl-beta-D-maltoside. The polymerase activity was made dependent upon exogenous RNA by destruction of the endogenous template RNA with micrococcal nuclease. Addition of BBV RNA1 or RNA2 induced synthesis of full-length negative-strand RNA isolated as a double-stranded complex with the added RNA. Newly synthesized plus strands were also detected in the RNA2 complexes. Certain other viral RNAs also induced synthesis of their negative strands.
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PMID:Template-dependent RNA polymerase from black beetle virus-infected Drosophila melanogaster cells. 241 18

Cytoplasmic extracts made from HeLa cells that have been harvested late after infection with vaccinia virus are capable of specifically transcribing templates containing vaccinia virus late-gene promoters. We applied such an extract to a phosphocellulose column and eluted the proteins with a series of buffers containing successively higher concentrations of NaCl. None of three column fractions alone was capable of specific transcription of a late-gene template. However, specific transcriptase activity could be reconstituted by mixing column fractions, with maximal activity seen when all three fractions were present. The activities present in all fractions were heat labile, resistant to micrococcal nuclease, and present only in extracts from vaccinia virus-infected cells. A quantitative complementation assay was used to further purify one factor, named VLTF-1, over subsequent columns of DEAE-cellulose and hydroxylapatite. VLTF-1 was separated from endogenous RNA polymerase, was a late-promoter-specific transcription factor, and had a sedimentation rate consistent with an apparent Mr of 45,000. The RNA polymerase-containing fraction was not only necessary for transcription with a late-promoter template but alone was capable of specifically transcribing a vaccinia virus early-gene promoter. A further difference between early and late gene transcription in this system was in the ability of the ATP analog beta-8-imidoadenosine-5'-triphosphate (AMP-PNP) to substitute for ATP in supporting specific transcription of only the late-promoter template. The system reconstituted from the various fractions retained the ability to produce the novel poly(A) sequence found on the 5' end of vaccinia virus late messages.
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PMID:Identification of factors specific for transcription of the late class of vaccinia virus genes. 247 68

35 S rRNA is the major intracellular precursor to 18, 5.8, and 25 S rRNAs in Saccharomyces cerevisiae. In this report, we show that the 3' termini of 35 S rRNA as well as 25 S rRNA are generated by post-transcriptional RNA processing rather than transcription termination. Using a partially purified yeast whole cell extract, efficient site-specific cleavage of a synthetic rRNA precursor was demonstrated in vitro. The 3' termini of the processed precursor were established by S1 nuclease protection analysis. RNA molecules containing the mature 3' termini of 35 and 25 S rRNA as well as molecules with a 3' terminus located 12 nucleotides beyond the 3' terminus of 25 S rRNA were the major products of the in vitro processing reaction. Processing activity required Mg2+ but was independent of ribonucleotides. Pretreatment of the yeast whole cell extract with proteinase K abolished processing activity, whereas micrococcal nuclease pretreatment of the extract had no effect on processing activity. These results show that RNA polymerase I-dependent transcription of yeast ribosomal cistrons continues beyond sequences that encode the 3' terminus of 35 S rRNA into the spacer region that separates 35 S rRNA transcription units.
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PMID:In vitro RNA processing generates mature 3' termini of yeast 35 and 25 S ribosomal RNAs. 264 84

Picornavirus RNAs are uncapped messengers and have unusually long 5' nontranslated regions (5'NTRs) which contain many noninitiating AUG triplets. The translational efficiency of different picornavirus RNAs varies between different cell-free extracts and even in the same extract, such as micrococcal nuclease-treated rabbit reticulocyte lysates. The effect of the poliovirus 5'NTR on in vitro translation was compared with that of the 5'NTR of encephalomyocarditis virus by the use of synthetic mRNAs, micrococcal nuclease-treated HeLa cell extracts, and rabbit reticulocyte lysates. Artificial mono- and dicistronic mRNAs synthesized with T7 RNA polymerase were used to investigate whether the 5'NTR of encephalomyocarditis virus RNA contains a potential internal ribosomal entry site. The sequence between nucleotides 260 and 484 in the 5'NTR of encephalomyocarditis RNA was found to play a critical role in the efficient translation in both mono- and dicistronic mRNAs. Our data suggest that an internal ribosomal entry site resides in this region.
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PMID:A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. 283 90


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