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)

We have developed a simple procedure for isolating a transcriptional extract from whole yeast cells which obviates the requirement for nuclear isolation. Detection of accurate mRNA initiation by RNA polymerase II in the extract requires the use of a sensitive assay, recently described by Kornberg and co-workers (Lue, N. F., Flanagan, P. M., Sugimoto, K., and Kornberg, R. D. (1989) Science 246, 661-664) that involves activation by a GAL4-VP16 fusion protein and a template lacking guanosine residues in the coding strand. The extract is prepared from fresh or frozen yeast cells by disruption with glass beads and fractionation of proteins by ammonium sulfate precipitation. The alpha-amanitin-sensitive transcripts synthesized in the assay were identical to those produced in a parallel assay using a yeast nuclear extract. The activity of the whole cell extract is lower per mg of protein than a nuclear extract but proportional to the volume of the nucleus relative to the whole cell. The optimal ranges for several reaction components including template, mono- and divalent cations, and nucleotide substrate concentration were determined. Under optimal conditions the whole cell extract produced a maximum of approximately 1 X 10(-2) transcripts/template molecule in 30 min.
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PMID:Accurate initiation by RNA polymerase II in a whole cell extract from Saccharomyces cerevisiae. 218 68

The large subunit of RNA polymerase II contains a highly conserved and essential heptapeptide repeat (Pro-Thr-Ser-Pro-Ser-Tyr-Ser) at its carboxy terminus. Saccharomyces cerevisiae cells are inviable if their RNA polymerase II large subunit genes encode fewer than 10 complete heptapeptide repeats; if they encode 10 to 12 complete repeats cells are temperature-sensitive and cold-sensitive, but 13 or more complete repeats will allow wild-type growth at all temperatures. Cells containing C-terminal domains (CTDs) of 10 to 12 complete repeats are also inositol auxotrophs. The phenotypes associated with these CTD mutations are not a consequence of an instability of the large subunit; rather, they seem to reflect a functional deficiency of the mutant enzyme. We show here that partial deletion mutations in RNA polymerase II CTD affect the ability of the enzyme to respond to signals from upstream activating sequences in a subset of promoters in yeast. The number of heptapeptide repeats required for maximal response to signals from these sequences differs from one upstream activating sequence to another. One of the upstream elements that is sensitive to truncations of the CTD is the 17-base-pair site bound by the GAL4 transactivating factor.
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PMID:RNA polymerase II C-terminal repeat influences response to transcriptional enhancer signals. 221 64

Fusion of the DNA-binding domain of yeast GAL4 protein to the amino terminus of bacteriophage T7 RNA polymerase yields a chimera that retains the characteristics of its components. The presence of the GAL4 peptide allows the chimeric enzyme to anchor itself on the DNA template, and this anchoring in turn drives the formation of a supercoiled DNA loop, in linear or circular templates, when RNA synthesis at the polymerase site forces a translocation of the DNA relative to the site. Nonspecific interaction between the chimeric enzyme and DNA appears to be sufficient to effect supercoiling during transcription. Transcription by the chimeric polymerase is strictly dependent on the presence of a T7 promoter; thus it provides a tool in vitro and in vivo for specifically supercoiling DNA segments containing T7 promoter sequences.
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PMID:Template supercoiling by a chimera of yeast GAL4 protein and phage T7 RNA polymerase. 239 63

The activation domains of eukaryotic DNA-binding transcription factors, such as GAL4, may regulate transcription by contacting RNA polymerase II. One potential site on RNA polymerase II for such interactions is the C-terminal tandemly repeated heptapeptide domain in the largest subunit (RPO21). We have changed the number of heptapeptide repeats in this yeast RPO21 C-terminal domain and have expressed these mutant RNA polymerase II polypeptides in yeast cells containing either wild-type or defective GAL4 proteins. Although the number of RPO21 heptapeptide repeats had no effect on the activity of wild-type GAL4, changing the length of the C-terminal domain modified the ability of mutant GAL4 proteins to activate transcription. Shorter or longer RPO21 C-terminal domains enhanced or partially suppressed, respectively, the effects of deletions in the transcriptional-activation domains of GAL4. The same RPO21 mutations also affected transcriptional activation by a GAL4-GCN4 chimera. These data suggest that the activation domains of DNA-binding transcription factors could interact, either directly or indirectly, with the heptapeptide repeats of RNA polymerase II.
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PMID:Mutations in RNA polymerase II enhance or suppress mutations in GAL4. 249 35

Ribosomal gene transcription requires the functional interplay of at least two promoter elements, the upstream control element (UCE) and the start site proximal core, which operate in concert to promote efficient and accurate transcription initiation by RNA polymerase I (pol I). Because this bipartite organization of the rDNA promoter is formally analogous to the organization of a typical pol II promoter, we have examined whether transcriptional activation by upstream activating sequences is brought about by similar molecular mechanisms for both classes of genes. We have replaced the UCE of the mouse rDNA promoter by three different pol II activating sequences (the yeast GAL4 binding sites, the target sequence of the enhancer binding protein E2 from bovine papilloma virus type 1 and the octamer motif), and measured the template activity of these chimeric promoters in the presence of the trans-activating proteins either in a cell free transcription system or in vivo after transfection into mouse cells. In the context of the pol I promoter none of these transcriptional activators enhanced rDNA transcription. The results indicate that activation by UCEs is not interchangeable between genes transcribed by RNA pol I and II, respectively, and suggest that different molecular mechanisms mediate the synergistic action of distant control sequences of different classes of genes.
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PMID:Transcriptional enhancement by upstream activators is brought about by different molecular mechanisms for class I and II RNA polymerase genes. 258 92

We have examined the effect of RNA polymerase II-dependent transcription on recombination between directly repeated sequences of the GAL10 gene in S. cerevisiae. Direct repeat recombination leading either to plasmid loss or conversion was examined in isogenic strains containing null mutations in the positive activator, GAL4, or the repressor, GAL80. A 15-fold increase in the rate of plasmid loss is observed in cells constitutively expressing the construct compared with cells that are not. Conversion events that retain the integrated plasmid are not stimulated by expression of the repeats. Northern analysis of strains containing plasmid inserts with various promoter mutations suggests that the stimulation in recombination is mediated by events initiating within the integrated plasmid sequences.
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PMID:Elevated recombination rates in transcriptionally active DNA. 264 56

The recent discovery that the activation domains of transcriptional activators (e.g., GAL4) from a number of species are interchangeable has led to the concept of a general mechanism for activation of RNA polymerase II genes. We have examined the different activities of the SV40 octamer motif ATGCAAAG in B cells and in HeLa cells in the context of either the beta-globin promoter, a TATA-box-containing mRNA promoter, or the U2 snRNA promoter, which contains a snRNA-specific proximal element. In the context of the beta-globin promoter, the octamer motif is a B-cell-specific enhancer element, whereas it is a ubiquitous enhancer element for the U2 snRNA promoter. The U2 promoter is unique in that it is not activated by enhancer elements that activate the beta-globin promoter, and a hybrid U2 promoter containing the upstream activating sequence UASG is not stimulated by a yeast GAL4 trans-activator. Together, these observations suggest that in the context of the U2 promoter, the octamer motif defines a new class of RNA polymerase II enhancer elements, which bind transcription factors that trans-activate gene expression by a different mechanism than the general mechanism mentioned above. These results are discussed in light of the possibility that the ubiquitous octamer binding protein Oct-1 and the B-cell-specific octamer binding protein Oct-2 are involved in the activation of the U2 and beta-globin promoters, respectively.
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PMID:Activation of the U2 snRNA promoter by the octamer motif defines a new class of RNA polymerase II enhancer elements. 285 85

GAL4 is a transcriptional activator found in yeast. Two distinct functions of the protein are required for its activity: one directs sequence-specific DNA binding, and another interacts with some other component of the transcriptional machinery, for example, RNA polymerase II or a TATA-binding protein. Two short regions of GAL4 function as 'activating sequences' when attached to the DNA-binding portion of GAL4 and these regions can be replaced by a large number of peptides encoded by Escherichia coli genomic DNA fragments or by a synthetic peptide designed to form an amphiphilic alpha-helix. All of these activating sequences, like that found in another yeast activator, GCN4 bear an excess negative charge. GAL4 and its derivatives that are active in yeast stimulate transcription in mammalian cells when GAL4 binding sites are introduced upstream of a mammalian gene; similarly, GAL4 activates transcription in Drosophila cells. Here we show that GAL4 derivatives stimulate gene expression in plant cells.
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PMID:Yeast activators stimulate plant gene expression. 316 94

Most class B (II) promoter regions from higher eukaryotes contain the TATA box and upstream and enhancer elements. Both the upstream and enhancer elements and their cognate factors have regulatory functions, whereas the TATA sequence interacts with the TATA box factor BTF1 to position RNA polymerase B and its ancillary initiation factors (STF, BTF2 and BTF3) to direct the initiation of transcription approximately 30 base pairs downstream. In many respects, class B promoter regions from the unicellular eukaryote Saccharomyces cerevisiae are similarly organized, containing upstream activating sequences that bear many similarities to enhancers. Although they are essential for initiation, the yeast TATA sequences are located at variable distances and further from the start sites (40-120 base pairs), whose locations are primarily determined by an initiator element. The basic molecular mechanisms that control initiation of transcription are known to be conserved from yeast to man: the yeast transcriptional transactivator GAL4 can activate a minimal TATA box-containing promoter in human HeLa cells, and a human inducible enhancer factor, the oestrogen receptor, can activate a similar minimal promoter in yeast. This striking evolutionary conservation prompted us to look for the presence in yeast of an activity that could possibly substitute for the human TATA box factor. We report here the existence of such an activity in yeast extracts.
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PMID:A yeast activity can substitute for the HeLa cell TATA box factor. 329 Jun 88

To distinguish between mechanisms of eukaryotic transcriptional activation, we tested whether yeast upstream promoter elements can stimulate transcription by a heterologous transcription machinery, bacteriophage T7 RNA polymerase. The gal enhancer-like element recognized by GAL4 protein or the ded1 poly(dA-dT) element was placed upstream of the T7 promoter and his3 structural gene, and T7 RNA polymerase was produced in yeast cells. Under conditions where the gal element would normally be either activating or nonactivating, his3 transcription by T7 RNA polymerase was not stimulated above the level observed in the absence of any upstream element. In contrast, the ded1 poly(dA-dT) element stimulated transcription 7-fold, similar to the enhancement observed on the native ded1 promoter. Activation by the ded1 element thus may involve effects on the chromatin template that facilitate entry of the transcription machinery, whereas activation by the gal element may involve specific contacts between GAL4 and the transcriptional machinery.
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PMID:Distinguishing between mechanisms of eukaryotic transcriptional activation with bacteriophage T7 RNA polymerase. 330 61


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