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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)

BTF3 is a human protein that is thought to be involved in transcription by RNA polymerase II [Zheng et al., Cell 50, 361-368, 1987]. A yeast homologue of BTF3, Egd1p, has been identified by its ability to enhance DNA binding of the Gal4p activator [Parthun et al., Mol. Cell. Biol. 12, 5683-5689, 1992]. We have cloned a second yeast gene, BTT1, which also encodes a BTF3 homologue. Btt1p and Egd1p are highly similar in sequence, which suggests that they are duplicated proteins with similar functions. Gene disruptions were used to investigate the function of the two proteins. Consistent with published results, we found that loss of EGD1 causes a minor defect in GAL gene induction. Loss of BTT1 has little if any effect. Surprisingly, we found that cells which lack both genes instead express the GAL1 and GAL10 mRNAs at much higher levels than wild type cells. This suggests that BTF3 really plays a negative role in GAL gene expression. Further experiments revealed that expression of the ACT1 and SSO1 genes also is elevated in cells that lack EGD1 and BTT1. In contrast, expression of rRNA and tRNA was not affected. We conclude that Btt1p and Egd1p have redundant functions in vivo, and that they exert a negative effect on the expression of several genes that are transcribed by RNA polymerase II.
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PMID:Yeast BTF3 protein is encoded by duplicated genes and inhibits the expression of some genes in vivo. 805 29

We have fused representatives of three structurally and functionally distinct classes of mammalian transcription activation domains for RNA polymerase II to the yeast GAL4 DNA binding domain. All fusion proteins were stable when expressed in yeast and were tested for their ability to activate transcription from various positions in the yeast GAL1 promoter. Activation domains functional from remote as well as TATA-proximal positions in mammalian cells, e.g. the acidic-type domain of VP16, also stimulate transcription in yeast from various promoter positions. Proline-rich domains, as e.g. in AP-2 and CTF/NF1, with considerable promoter activity and low enhancer activity in mammalian cells stimulate transcription in yeast only from a position close to the TATA box. The glutamine-rich domains of Oct1, Oct2 and Sp1, which activate transcription in mammalian cells from close to the TATA box in response to a remote enhancer, are inactive in the yeast GAL1 promoter. This finding might reflect some basic difference between the organization of yeast and mammalian promoters.
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PMID:Functional differences between mammalian transcription activation domains at the yeast GAL1 promoter. 831 9

Transcription-dependent DNA melting on the yeast GAL1 and GAL10 promoters was found to be more closely correlated with the TATA box than the transcription start site. On both these genes, melting begins about 20 base pairs downstream of the TATA box. Physical and genetic analyses suggest that RNA polymerase II associates with this region. Thus, the distance between promoter melting and the TATA box in yeast may be similar to that in higher eukaryotes, even though transcription initiates in a region about 10 to 90 base pairs farther downstream in yeast.
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PMID:DNA melting on yeast RNA polymerase II promoters. 834 41

The Saccharomyces cerevisiae GAL1 and GAL10 genes are controlled in response to the availability of galactose and glucose by multiple activating and repressing proteins bound at adjacent or overlapping sites in UASG. Negative control elements in UASG, designated GAL operators GALO1 to GALO6, are required to silence basal level transcription of GAL1 and GAL10 when galactose is absent. We isolated and characterized recessive mutations in six nuclear genes, TSF1 to TSF6, that impair silencing of GAL1 and GAL10 gene expression. Surprisingly, the results of several experiments suggest that the TSF genes encode global regulatory factors. tsf1 to tsf6 mutations derepressed expression from yeast CYC-GAL hybrid promoters (fused to lacZ) that harbor a variety of operator sequences, and caused pleiotropic defects in cell growth, mating, and sporulation. S1 mapping and Northern blot results for tsf3 suggest that the molecular defect is at the transcriptional level. Mutant phenotypes were additive in certain combinations of tsf double mutants, implying that more than one silencing pathway is involved in TSF1 to TSF6 function. Most significantly, mutations in all six TSF1 to TSF6 genes activated expression from GAL1 and CYC1 promoters (fused to lacZ) lacking upstream activating sequences. Combined, the simplest interpretation of these results is that TSF1 to TSF6 encode factors that control the function of the basic RNA polymerase II transcriptional machinery.
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PMID:TSF1 to TSF6, required for silencing the Saccharomyces cerevisiae GAL genes, are global regulatory genes. 834 4

Mutant a and alpha yeast cells were created with histone H3 containing cysteine in place of alanine 110. Because transcriptionally active nucleosomes "unfold" to reveal the histone H3-thiol groups at the center of the core, the active nucleosomes of the mutant strain can be isolated by mercury-affinity chromatography. We compared the unbound and mercury-bound nucleosomes of haploid H3-mutant strains expressing either the MAT alpha or the MATa mating-type locus. In a MAT alpha strain, the Hg-bound nucleosomes are enriched in MAT alpha DNA but lack the DNA of the transcriptionally silent HMRa mating-type locus. Conversely, in a MATa strain, the Hg-bound nucleosomes are enriched in MATa DNA sequences but deficient in HML alpha DNA. When the SIR3 gene, known to be required for silencing of the repressed mating-type loci, is mutated in the MAT alpha strain, transcription of the HMRa ensues, and its nucleosomes, as well as those of the MAT alpha locus, are retained by the organomercurial column. It follows that derepression of the silent mating-type locus, caused by the sir3 null mutation, is accompanied by an unfolding of its nucleosomes to reveal the histone H3 SH groups at their centers. Nucleosomes of the pheromone-encoding gene MFA2, a gene that is expressed in MATa cells but not in MAT alpha cells, are bound to the organomercurial column when isolated from MATa cells but not from MAT alpha cells. Thus, there is a good correlation between nucleosome unfolding and the renewed transcriptional activity at mating-type loci, and at MFA2, which had been silenced for prolonged periods. A close temporal correlation between nucleosome refolding and the cessation of transcription is not always observed in yeast, however, in contrast to observations in mammalian cells. For example, nucleosomes of the GAL1 gene are maintained in a "poised" or "primed" thiol-reactive state even when the gene is not being transcribed (Chen, T. A., Smith, M. M., Le, S., Sternglanz, R., and Allfrey, V. G. (1991) J. Biol. Chem. 266, 6489-6498). It follows that the unfolding of the nucleosome cores of the yeast H3 mutant is regulated by factors that are not temporally linked to the recruitment or traverse of the RNA polymerase complex, but which may determine the rate at which different domains of chromatin adapt to the need for transcription of the associated DNA sequences.
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PMID:Nucleosome structural changes during derepression of silent mating-type loci in yeast. 841 18

Despite evidence that DNA topoisomerase I is required to relieve torsional stress during DNA replication and transcription, yeast strains with a top1 null mutation are viable and display no gross defects in DNA or RNA synthesis, possibly because other proteins provide overlapping functions. We isolated mutants whose inviablility or growth defect is relieved when TOP1 is expressed [trf mutants (topoisomerase one-requiring function)]. The TRF genes define at least four complementation groups. TRF3 is allelic to TOP2. TRF1 is allelic to HPR1, previously shown to be homologous to TOP1 over two short regions. TRF4 encodes a novel 584-amino acid protein with homology to the N-terminus of Saccharomyces cerevisiae topo I. Like top1 mutants, trf4 mutants have elevated rDNA recombination and fail to shut off RNA polymerase II transcription in stationary phase. trf4 null mutants are cs for viability, display reduced expression of GAL1 and Cell Cycle Box UAS::LacZ fusions, and are inviable in combination with trfI null mutants, indicating that both proteins may share a common function with DNA topoisomerase I. The existence of multiple TRF complementation groups suggests that not all biological functions of topo I can be carried out by topo II.
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PMID:Isolation of mutants of Saccharomyces cerevisiae requiring DNA topoisomerase I. 864 85

Homologous recombination in Saccharomyces cerevisiae and other organisms can be stimulated by transcription. Consistent with this, we find that recombination of a chromosomal ade1 allele with a plasmid-borne ADE1 ORF under the control of the GAL1 promoter increased from 6.1x10(-6) to 1.7x10(-4) when transcription of the plasmid locus was induced by growing the cells in the presence of galactose. Recombination could also be stimulated by over-expressing the Gal4 transcription factor in the presence of the GAL1-ADE1 plasmid, while culturing the cells in dextrose medium. However, when transcription of the same ORF was driven from the highly active promoters of the rDNA (RNA polymerase I), and ADH1 (RNA polymerase II) genes, only background levels of recombination (5-10x10(-6)) were observed, irrespective of the carbon source. Recombination was found to involve integration of the whole plasmid and to depend on RAD51, RAD52 and RAD54. The results indicate that increased accessibility of transcriptionally active chromatin is not sufficient to cause increased rates of this kind of reciprocal exchange.
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PMID:Stimulation of mitotic recombination upon transcription from the yeast GAL1 promoter but not from other RNA polymerase I, II and III promoters. 892 89

Nonhistone proteins 6A and 6B (NHP6A/B) are nonsequence-specific DNA-binding proteins from Saccharomyces cerevisiae that are related structurally and functionally to the mammalian high mobility group proteins 1 and 2. These DNA architectural proteins distort DNA structure severely and have been shown to promote assembly of specialized recombination complexes. Here we show that the yeast NHP6A/B proteins are required for the induction of a subset of genes transcribed by RNA polymerase II (pol II). Activation of the CUP1, CYC1, GAL1, and DDR2 genes was decreased or abolished completely in the delta nhp6A/B strain. No significant change in basal expression was observed for any of the 10 genes examined. Analysis of chimeric gene constructs localized the regions dependent on NHP6A/B to be primarily at the core promoters, although the GAL1 UAS also requires NHP6A/B for activity. In vitro, NHP6A stimulated transcription by pol II at the GAL1 promoter three- to fivefold above the level of activation by GAL4-VP16 alone. Gel mobility shift assays showed that NHP6A promotes the formation of a complex with TBP and TFIIA at the TATA box that has enhanced affinity for TFIIB.
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PMID:Yeast HMG proteins NHP6A/B potentiate promoter-specific transcriptional activation in vivo and assembly of preinitiation complexes in vitro. 894 17

We examine transcriptional activation and chromatin remodeling at the PHO5 promoter in yeast by fusion proteins that are thought to act by recruiting the RNA polymerase II holoenzyme to DNA in the absence of a classic activating region. These hybrid proteins (e.g., Gal11+Pho4 or Gal4(58-97)+Pho4 in the presence of a GAL11P allele) efficiently activated transcription and remodeled chromatin. Similar chromatin remodeling was observed at a PHO5 promoter deleted for TATA and thus unable to support transcription. We conclude that recruitment of the holoenzyme or associated proteins suffices for chromatin remodeling. We also show that the SWI/SNF complex is required neither for efficient transcription of the wild-type PHO5 nor the GAL1 promoters, and we observe nearly complete chromatin remodeling at PHO5 in the absence of Snf2.
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PMID:RNA polymerase II holoenzyme recruitment is sufficient to remodel chromatin at the yeast PHO5 promoter. 909 14

A mutation in RPB5 (rpb5-9), an essential RNA polymerase subunit assembled into RNA polymerases I, II, and III, revealed a role for this subunit in transcriptional activation. Activation by GAL4-VP16 was impaired upon in vitro transcription with mutant whole-cell extracts. In vivo experiments using inducible reporter plasmids and Northern analysis support the in vitro data and demonstrate that RPB5 influences activation at some, but not all, promoters. Remarkably, this mutation maps to a conserved region of human RPB5 implicated by others to play a role in activation. Chimeric human-yeast RPB5 containing this conserved region now can function in place of its yeast counterpart. The defects noted with rpb5-9 are similar to those seen in truncation mutants of the RPB1-carboxyl terminal domain (CTD). We demonstrate that RPB5 and the RPB1-CTD have overlapping roles in activation because the double mutant is synthetically lethal and has exacerbated activation defects at the GAL1/10 promoter. These studies demonstrate that there are multiple activation targets in RNA polymerase II and that RPB5 and the CTD have similar roles in activation.
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PMID:RNA polymerase subunit RPB5 plays a role in transcriptional activation. 986 Sep 60


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