Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

U6 snRNA is the only spliceosomal snRNA transcribed by RNA polymerase III in yeast. We have constructed a regulated U6 snRNA transcription unit by introducing the binding site for the Escherichia coli lacI repressor protein in the U6 snRNA promoter. GAL-induced expression of lacI protein led to a decrease in U6 snRNA levels and blocked cell growth. lacI dissociation from the promoter, and consequent U6 snRNA transcription, could be induced by addition of IPTG and repression of lacI transcription. To test the usefulness of this system in studying spliceosomal U6 snRNA function, we conditionally expressed U6 snRNAs with a single base substitution in position A51. We demonstrate that expression of the U6-A51 mutations confers a strong dominant negative phenotype as shown by severe reductions in growth rate. In these strains, splicing of endogenous pre-mRNAs was blocked before the second step.
 ...
PMID:Construction of an in vivo-regulated U6 snRNA transcription unit as a tool to study U6 function. 957 Mar 23

The mediator complex is essential for regulated transcription in vitro. In the yeast Saccharomyces cerevisiae, mediator comprises >15 subunits and interacts with the C-terminal domain of the largest subunit of RNA polymerase II, thus forming an RNA polymerase II holoenzyme. Here we describe the molecular cloning of the MED1 cDNA encoding the 70-kDa subunit of the mediator complex. Yeast cells lacking the MED1 gene are viable but show a complex phenotype including partial defects in both repression and induction of the GAL genes. Together with results on other mediator subunits, this implies that the mediator is involved in both transcriptional activation and repression. Similar to mutations in the SRB10 and SRB11 genes encoding cyclin C and the cyclin C-dependent kinase, a disruption of the MED1 gene can partially suppress loss of the Snf1 protein kinase. We further found that a lexA-Med1 fusion protein is a strong activator in srb11 cells, which suggests a functional link between Med1 and the Srb10/11 complex. Finally, we show that the Med2 protein is lost from the mediator on purification from Med1-deficient cells, indicating a physical interaction between Med1 and Med2.
 ...
PMID:The Med1 subunit of the yeast mediator complex is involved in both transcriptional activation and repression. 989 41

Phosphorylation of the yeast transcription factor GAL4 at S699 is required for efficient galactose-inducible transcription. We demonstrate that this site is a substrate for the RNA polymerase holoenzyme-associated CDK SRB10. S699 phosphorylation requires SRB10 in vivo, and this site is phosphorylated by purified SRB10/ SRB11 CDK/cyclin in vitro. RNA Pol II holoenzymes purified from WT yeast phosphorylate GAL4 at sites observed in vivo whereas holoenzymes from srb10 yeast are incapable of phosphorylating GAL4 at S699. Mutations at GAL4 S699 and srb10 are epistatic for GAL induction, demonstrating that SRB10 regulates GAL4 activity through this phosphorylation in vivo. These results demonstrate a function for the SRB10/ CDK8 holoenzyme-associated CDK that involves regulation of transactivators by phosphorylation during transcriptional activation.
 ...
PMID:GAL4 is regulated by the RNA polymerase II holoenzyme-associated cyclin-dependent protein kinase SRB10/CDK8. 1036 Jan 83

Cyclin C and the cyclin C-dependent protein kinase are associated with the RNA polymerase II Mediator complex, which regulates initiation of transcription in response to signals from activators and repressors bound to upstream promoter elements. Disruption of the corresponding genes, SRB11 and SRB10, in budding yeast causes a reduction in expression of the GAL genes, which is particularly pronounced in a mig1 snf1 background. We have screened two yeast genomic libraries for genes that can suppress this phenotype when overexpressed. Seven suppressor genes were identified, GIS1-7. GIS1 encodes one of two related zinc-finger proteins, which also share two other highly conserved domains present in several eukaryotic transcription factors. GIS2 encodes a homologue of the mammalian CNBP and fission yeast Byr3 proteins. GIS3 and GIS4 predict proteins with no obvious similarities to any known proteins. GIS5-7 are identical to the previously described genes PDE2, SGE1 and TUB3, respectively. None of the suppressor genes seem to be involved in Mediator function. Instead, we find that the GIS1, GIS2 and GIS4 genes interact with the CDC25 gene, indicating a possible involvement of these genes in the RAS/cAMP signaling pathway.
 ...
PMID:Yeast genes GIS1-4: multicopy suppressors of the Gal- phenotype of snf1 mig1 srb8/10/11 cells. 1062 41

The general transcription factor TFIIH is required for initial DNA unwinding and promoter escape by RNA polymerase II in vitro. We examined whether Rad25p, a DNA helicase subunit of TFIIH, mediates promoter opening and promoter escape in the yeast Saccharomyces cerevisiae. DNA unwinding was probed with an in vivo permanganate reactivity assay, in a temperature-sensitive mutant of RAD25. The consequences of Rad25p inactivation were promoter-specific. Whereas in the TDH2 promoter permanganate reactivity was entirely abolished, the reactivity at the GAL1 and GAL10 promoter regions was only moderately affected. In the GAL genes permanganate reactivity uniformly decreased downstream of the transcription start site, indicating that progression of RNA polymerase II to this region was impaired. Our results suggest that in yeast cells, promoter opening is not sufficient for productive initiation and that Rad25p-mediated promoter escape may be a limiting step in the transcription of some promoters.
 ...
PMID:Rad25p, a DNA helicase subunit of yeast transcription factor TFIIH, is required for promoter escape in vivo. 1071 51

Gal4p activates transcription of the Saccharomyces GAL genes in response to galactose and is phosphorylated during interaction with the RNA polymerase II (Pol II) holoenzyme. One phosphorylation at S699 is necessary for full GAL induction and is mediated by Srb10p/CDK8 of the RNA Pol II holoenzyme mediator subcomplex. Gal4p S699 phosphorylation is necessary for sensitive response to inducer, and its requirement for GAL induction can be abrogated by high concentrations of galactose in strains expressing wild-type GAL2 and GAL3. Gal4p S699 phosphorylation occurs independently of Gal3p and is responsible for the long-term adaptation response observed in gal3 yeast. SRB10 and GAL3 are shown to represent parallel mechanisms for GAL gene induction. These results demonstrate that Gal4p activity is controlled by two independent signals: one that acts through Gal3p-galactose and a second that is mediated by the holoenzyme-associated cyclin-dependent kinase Srb10p. Since Srb10p is regulated independently of galactose, our results suggest a function for CDK8 in coordinating responses to specific inducers with the environment through the phosphorylation of gene-specific activators.
 ...
PMID:Multiple signals regulate GAL transcription in yeast. 1080 31

An in vivo protein interaction assay was used to search a yeast cDNA library for proteins that bind to the acidic activation domain (AD) of the yeast Gal4 protein. Sug2 protein, a component of the 19 S regulatory particle of the 26 S proteasome, was one of seven proteins identified in this screen. In vitro binding assays confirm a direct interaction between these proteins. SUG2 and SUG1, another 19 S component, were originally discovered as a mutation able to suppress the phenotype of a Gal4 truncation mutant (Gal4(D)p) lacking much of its AD. Sug1p has previously been shown to bind the Gal4 AD in vitro. Taken together, these genetic and biochemical data suggest a biologically significant interaction between the Gal4 protein and the 19 S regulatory particle of the proteasome. Indeed, it is demonstrated here that the Gal4 AD interacts specifically with immunopurified 19 S complex. The proteasome regulatory particle has been shown recently to play a direct role in RNA polymerase II transcription and the activator-19 S interaction could be important in recruiting this large complex to transcriptionally active GAL genes.
 ...
PMID:The Gal4 activation domain binds Sug2 protein, a proteasome component, in vivo and in vitro. 1141 96

Evolutionarily conserved variant histone H2A.Z has been recently shown to regulate gene transcription in Saccharomyces cerevisiae. Here we show that loss of H2A.Z in this organism negatively affects the induction of GAL genes. Importantly, fusion of the H2A.Z C-terminal region to S phase H2A without its corresponding C-terminal region can mediate the variant histone's specialized function in GAL1-10 gene induction, and it restores the slow-growth phenotype of cells with a deletion of HTZ1. Furthermore, we show that the C-terminal region of H2A.Z can interact with some components of the transcriptional apparatus. In cells lacking H2A.Z, recruitment of RNA polymerase II and TATA-binding protein to the GAL1-10 promoters is significantly diminished under inducing conditions. Unexpectedly, we also find that H2A.Z is required to globally maintain chromatin integrity under GAL gene-inducing conditions. We hypothesize that H2A.Z can positively regulate gene transcription, at least in part, by modulating interactions with RNA polymerase II-associated factors at certain genes under specific cell growth conditions.
 ...
PMID:H2A.Z is required for global chromatin integrity and for recruitment of RNA polymerase II under specific conditions. 1150 69

The basic helix-loop-helix (bHLH) transcription factor, Hand1, plays an important role in the development of the murine extra-embryonic trophoblast cell lineage. In the present study, we have analysed the expression of Hand1 in human extra-embryonic cell types and determined its binding specificity and transcriptional activity upon interaction with different class A bHLH factors. Northern blotting and in situ hybridization showed that Hand1 mRNA is specifically expressed in amnion cells at different stages of gestation. Accordingly, we demonstrate that the protein is exclusively produced in the amniotic epithelium in vivo and in purified amnion cells in vitro using a novel polyclonal Hand1 antiserum. Reverse transcriptase-PCR and immunohistochemical staining of blastocysts revealed the production of Hand1 mRNA and polypeptide in the trophectodermal cell layer. In the presence of E12/E47, Hand1 stimulated the transcription of luciferase reporters harbouring degenerate E-boxes, suggesting that E-proteins are potential dimerization partners in trophoblastic tumour and amnion cells. In contrast, Hand1 diminished E12/E47-dependent transcription of reporters containing perfect E-boxes by inhibiting the interaction of Hand1/E-protein heterodimers with the palindromic cognate sequence. Furthermore, we show that Hand1 down-regulated GAL-E12-dependent reporter expression, indicating that the protein can also act directly as a transcriptional repressor. Mutational analyses of GAL-Hand1 suggested that two protein regions located within its N-terminal portion mainly confer the repressing activity. In conclusion, human Hand1 may play an important role in the differentiation of the amniotic membrane and the pre-implanting trophoblast. Furthermore, the data suggest that Hand1 can act as a repressor by two independent mechanisms; sequestration of class A bHLH factors from E-boxes and inhibition of their transcriptional activity.
 ...
PMID:Human Hand1 basic helix-loop-helix (bHLH) protein: extra-embryonic expression pattern, interaction partners and identification of its transcriptional repressor domains. 1180 95

Initiation of transcription of protein-encoding genes by RNA polymerase II was thought to require transcription factor TFIID, a complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs). In the presence of TBP-free TAF complex (TFTC), initiation of polymerase II transcription can occur in the absence of TFIID. TFTC contains several subunits that have been shown to play the role of transcriptional coactivators, including the GCN5 histone acetyltransferase (HAT), which acetylates histone H3 in a nucleosomal context. Here we analyze the coactivator function of TFTC. We show direct physical interactions between TFTC and the two distinct activation regions (H1 and H2) of the VP16 activation domain, whereas the HAT-containing coactivators, p300/CBP (CREB-binding protein), interact only with the H2 subdomain of VP16. Accordingly, cell transfection experiments demonstrate the requirement of both p300 and TFTC for maximal transcriptional activation by GAL-VP16. In agreement with this finding, we show that in vitro on a chromatinized template human TFTC mediates the transcriptional activity of the VP16 activation domain in concert with p300 and in an acetyl-CoA-dependent manner. Thus, our results suggest that these two HAT-containing co-activators, p300 and TFTC, have complementary rather than redundant roles during the transcriptional activation process.
 ...
PMID:TATA-binding protein-free TAF-containing complex (TFTC) and p300 are both required for efficient transcriptional activation. 1210 88


<< Previous 1 2 3 4 Next >>