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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.
Mol Cell Biol 1990 May
PMID:RNA polymerase II subunit composition, stoichiometry, and phosphorylation. 218 13

We developed a procedure to measure mRNA decay rates in the yeast Saccharomyces cerevisiae and applied it to the determination of half-lives for 20 mRNAs encoded by well-characterized genes. The procedure utilizes Northern (RNA) or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpb1-1 temperature-sensitive mutant. We compared the results of this procedure with results obtained by two other procedures (approach to steady-state labeling and inhibition of transcription with Thiolutin) and also evaluated whether heat shock alter mRNA decay rates. We found that there are no significant differences in the mRNA decay rates measured in heat-shocked and non-heat-shocked cells and that, for most mRNAs, different procedures yield comparable relative decay rates. Of the 20 mRNAs studied, 11, including those encoded by HIS3, STE2, STE3, and MAT alpha 1, were unstable (t1/2 less than 7 min) and 4, including those encoded by ACT1 and PGK1, were stable (t1/2 greater than 25 min). We have begun to assess the basis and significance of such differences in the decay rates of these two classes of mRNA. Our results indicate that (i) stable and unstable mRNAs do not differ significantly in their poly(A) metabolism; (ii) deadenylation does not destabilize stable mRNAs; (iii) there is no correlation between mRNA decay rate and mRNA size; (iv) the degradation of both stable and unstable mRNAs depends on concomitant translational elongation; and (v) the percentage of rare codons present in most unstable mRNAs is significantly higher than in stable mRNAs.
Mol Cell Biol 1990 May
PMID:Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae. 218 28

Regulation of eukaryotic genes is largely governed by multiple cis-acting DNA sequences recognized by specific transcription factors. The transcription factor NF-kappa B has been implicated as an important regulator of cellular and viral genes, including those of immunoglobulin kappa light chain, interleukin-2, beta-interferon, HIV-1 and cytomegalovirus. We have analyzed the effect of increasing the number of NF-kappa B sites, located directly upstream from the TATA box. Four copies of the sequence gave a more than 100-fold stimulation relative to a single copy, suggesting that NF-kappa B proteins act synergistically to bring about this dramatic increase in transcription. By DNase I footprinting we demonstrated factor binding to two adjacent NF-kappa B sites in vitro. However, we found no evidence for co-operative binding to these DNA sites. We propose that the high transcriptional activity results from another type of co-operation, based on multiple weak interactions of the NF-kappa B factors with another component of the transcription apparatus, perhaps RNA polymerase II itself.
J Mol Biol 1990 Jul 20
PMID:Synergistic activation of transcription by multiple binding sites for NF-kappa B even in absence of co-operative factor binding to DNA. 219 80

REB1 is a DNA-binding protein that recognizes sites within both the enhancer and the promoter of rRNA transcription as well as upstream of many genes transcribed by RNA polymerase II. We report here the cloning of the gene for REB1 by screening a yeast genomic lambda gt11 library with specific oligonucleotides containing the REB1 binding site consensus sequence. The REB1 gene was sequenced, revealing an open reading frame encoding 809 amino acids. The predicted protein was highly hydrophilic, with numerous OH-containing amino acids and glutamines, features common to many of the general DNA-binding proteins of Saccharomyces cerevisiae, such as ABF1, RAP1, GCN4, and HSF1. There was some homology between a portion of REB1 and the DNA-binding domain of the oncogene myb. REB1 is an essential gene that maps on chromosome II. However, the physiological role that it plays in the cell has yet to be established.
Mol Cell Biol 1990 Oct
PMID:REB1, a yeast DNA-binding protein with many targets, is essential for growth and bears some resemblance to the oncogene myb. 220 8

Enhancers stimulate transcription of RNA polymerase II-transcribed genes in an orientation-independent manner and over long distances. This stimulation is known to be associated with an increased polymerase density over the linked gene. However, many aspects of the exact mechanism of remote gene control remain to be elucidated. Based on some reports on RNA polymerase I transcription, we wanted to test whether RNA polymerase II enters at the enhancer and from there proceeds towards the promoter while synthesizing unstable transcripts ("scanning/readthrough transcription" model). For this, we have inserted a complete terminator region from the mouse beta-globinmaj gene between the SV40 enhancer and the rabbit beta-globin promoter. In contrast to what the model predicts, insertion of the terminator had no affect on remote enhancer action. Furthermore, we have determined the RNA polymerase density over the spacer DNA between enhancer and promoter, and over the reporter gene, by means of the so-called run-on transcription assay. We find very low transcription of the spacer, but high transcription of the globin reporter gene. Thus, our data are not consistent with a scanning/readthrough transcription mechanism where RNA polymerase II would move from the enhancer to the promoter while transcribing the intervening spacer DNA. These and other findings are compatible with a model where enhancer and promoter are brought into close proximity, perhaps with concomitant looping out of the intervening DNA.
Somat Cell Mol Genet 1990 Jul
PMID:A transcriptional terminator between enhancer and promoter does not affect remote transcriptional control. 221 23

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.
Mol Cell Biol 1990 Nov
PMID:In vitro analysis of a transcription termination site for RNA polymerase II. 223 18

RNA polymerase II (RNAPII) is a complex multisubunit enzyme responsible for the synthesis of pre-mRNA in eucaryotes. The enzyme is made of two large subunits associated with at least eight smaller polypeptides, some of which are common to all three RNA polymerase species. We have initiated a genetic analysis of RNAPII by introducing mutations in RPO21, the gene encoding the largest subunit of RNAPII in Saccharomyces cerevisiae. We have used a yeast genomic library to isolate plasmids that can suppress a temperature-sensitive mutation in RPO21 (rpo21-4), with the goal of identifying gene products that interact with the largest subunit of RNAPII. We found that increased expression of wild-type RPO26, a single-copy, essential gene encoding a 155-amino-acid subunit common to RNAPI, RNAPII, and RNAPIII, suppressed the rpo21-4 temperature-sensitive mutation. Mutations were constructed in vitro that resulted in single amino acid changes in the carboxy-terminal portion of the RPO26 gene product. One temperature-sensitive mutation, as well as some mutations that did not by themselves generate a phenotype, were lethal in combination with rpo21-4. These results support the idea that the RPO26 and RPO21 gene products interact.
Mol Cell Biol 1990 Dec
PMID:A suppressor of an RNA polymerase II mutation of Saccharomyces cerevisiae encodes a subunit common to RNA polymerases I, II, and III. 224 52

RNA synthesis was stimulated directly in a cell-free expression system by crude preparations of recombinant mouse estrogen receptor (ER). Receptor-stimulated transcription required the presence of estrogen response elements (EREs) in the test template and could be specifically inhibited by addition of competitor oligonucleotides containing EREs. Moreover, polyclonal antibodies directed against the DNA-binding region of ER inhibited ER-dependent transcription. In our cell-free expression system, hormone-free ER induced transcription in a hormone-independent manner. Evidence is presented suggesting that ER acts by facilitating the formation of a stable preinitiation complex at the target gene promoter and thus augments the initiation of transcription by RNA polymerase II. These observations lend support to our current understanding of the mechanism of steroid receptor-regulated gene expression and suggest strong conservation of function among members of the steroid receptor superfamily.
Mol Cell Biol 1990 Dec
PMID:Mechanism of estrogen receptor-dependent transcription in a cell-free system. 224 75

Genomic and cDNA clones homologous to the RpII215 gene of Drosophila were isolated from Arabidopsis thaliana and assigned to a single copy gene encoding a transcript of 6.8 kb. Nucleotide sequence analysis of Arabidopsis genomic and cDNAs revealed a striking homology to yeast, Caenorhabditis, Drosophila and mouse genes encoding the largest subunit of RNA polymerase II. The Arabidopsis gene rpII215 contains 13 introns, 12 of which interrupt the coding sequence of a protein of 205 kDa. The position of the first intron is conserved between plant and animal genes, while an intron located in the 3' untranslated region of the rpII215 gene is unique to Arabidopsis. Common domains present in all known largest subunits of eucaryotic RNA polymerase II were identified in the predicted sequence of the Arabidopsis RpII215 protein. Both the order and the position of N-terminal Zn2+ finger and of DNA and alpha-amanitin binding motifs are conserved in Arabidopsis. The C-terminal region of the Arabidopsis protein contains 15 consensus and 26 variant YSPTSPS repeats (CTDs). Highly conserved structure among the various C-terminal domains suggests that the largest subunit of RNA polymerase II in plants may also interact with transcription factors and with protein kinases that control the cell cycle as in other organisms.
Mol Gen Genet 1990 Aug
PMID:Homologous domains of the largest subunit of eucaryotic RNA polymerase II are conserved in plants. 225 44

To investigate the effect of ligand (be it hormone, antihormone, or no hormone) on the interaction between estrogen receptor (ER) and chromatin, we have used formaldehyde as a cross-linking agent in intact MCF-7 human breast cancer cells. After a 1- to 2-h hormone treatment, the cells are exposed for 8 min to formaldehyde, which is added directly to their culture medium to minimize environmental perturbation. Nuclei are prepared from formaldehyde-treated cells and their contents are fractionated on CsCl density gradients to separate DNA-protein complexes from free protein. Peak gradient fractions are assayed for the presence of specific proteins by immunoblot of sodium dodecyl sulfate-polyacrylamide gel patterns. Using this approach, we find that 0.15% formaldehyde is optimal for cross-linking ER to chromatin. We detect ER and the large subunit of RNA polymerase II with DNA from formaldehyde-treated, but not from untreated cells. On the other hand, actin (a cytoplasmic protein) and small nuclear ribonucleoprotein particle proteins (nuclear RNA binding proteins) are not cross-linked to DNA. Therefore, cross-linking appears to be selective and fractionation is efficient. Interestingly, we detect similar levels of ER (as well as RNA polymerase II) with DNA from formaldehyde-treated cells, regardless of whether the cells are preexposed to estrogen (17 beta-estradiol at 10(-8) M), antiestrogen (ICI 164,384 at 10(-7) or 10(-6) M), or no hormone. These results, using covalent cross-linking in intact cells, indicate that both ligand-occupied and unoccupied ER are associated with chromatin.
Mol Endocrinol 1990 Nov
PMID:Cross-linking of estrogen receptor to chromatin in intact MCF-7 human breast cancer cells: optimization and effect of ligand. 228 Jul 70


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