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PubMed1995

Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III-dependent small nuclear RNA genes.

The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP-TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific as gene-specific factors for PSE-and B-box-containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the from used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.

Base Sequence↗
PubMed1998

The largest subunit of mouse RNA polymerase II (RPB1) functionally substituted for its yeast counterpart in vivo.

The full-length mouse RNA polymerase II (pol II) largest subunit (RPB1) gene was used to replace 5070 bp of the yeast Saccharomyces cerevisiae RPB1 gene via homologous recombination and gene replacement in vivo. Transcription of the mouse RPB1 gene using the yeast promoter in the haploid state was confirmed by Northern analysis. This strain of yeast is viable, indicating that mouse RPB1 is able to interact functionally with the other yeast RNA pol II subunits in vivo.

Animals↗
PubMed2002

Human CRSP interacts with RNA polymerase II CTD and adopts a specific CTD-bound conformation.

Activation of gene transcription in mammalian cells requires several classes of coactivators that participate in different steps of the activation cascade. Using conventional and affinity chromatography, we have isolated a human coactivator complex that interacts directly with the C-terminal domain (CTD) of RNA polymerase II (Pol II). The CTD-binding complex is structurally and functionally indistinguishable from our previously isolated CRSP coactivator complex. The closely related, but transcriptionally inactive, ARC-L complex failed to interact with the CTD, indicating a significant biochemical difference between CRSP and ARC-L that may, in part, explain their functional divergence. Electron microscopy and three-dimensional single-particle reconstruction reveals a conformation for CTD-CRSP that is structurally distinct from unliganded CRSP or CRSP bound to SREBP-1a, but highly similar to CRSP bound to the VP16 activator. Together, our findings suggest that the human CRSP coactivator functions, at least in part, by mediating activator-dependent recruitment of RNA Pol II via the CTD.

Binding Sites↗
PubMed1995

Complex patterns of transcription of a Drosophila retrotransposon in vivo and in vitro by RNA polymerases II and III.

The mdg1 retrovirus-like retrotransposon of Drosophila melanogaster was found to possess a complex promoter which can be transcribed by both RNA polymerases II and III (pol II and pol III). Pol III transcription, which is not typical of protein-coding genes, is driven by the sequences located in the long terminal repeat (LTR) of mdg1, predominantly within the transcribed region and is initiated 10 bp upstream from the regular pol II RNA start site. The pol III RNA start site is observed not only in in vitro transcription reactions, but also in total RNA isolated from tissue culture cells, larvae, pupae and adult flies. A possible role of pol III transcription in mechanisms controlling the expression of full-length mdg1-encoded transcripts in the developing fly, which are apparently relaxed in cell culture, is discussed.

Amanitins↗
PubMed2000

DSIF and NELF interact with RNA polymerase II elongation complex and HIV-1 Tat stimulates P-TEFb-mediated phosphorylation of RNA polymerase II and DSIF during transcription elongation.

Control of transcription elongation requires a complex interplay between the recently discovered positive transcription elongation factor b (P-TEFb) and negative transcription elongation factors, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) sensitivity inducing factors (DSIF) and the negative elongation factor (NELF). Activation of HIV-1 gene expression is regulated by a nascent RNA structure, termed TAR RNA, in concert with HIV-1 Tat protein and these positive and negative elongation factors. We have used a stepwise RNA pol II walking approach and Western blotting to determine the dynamics of interactions between HIV-1 Tat, DSIF/NELF, and the transcription complexes actively engaged in elongation. In addition, we developed an in vitro kinase assay to determine the phosphorylation status of proteins during elongation stages. Our results demonstrate that DSIF/NELF associates with RNA pol II complexes during early transcription elongation and travels with elongation complexes as the nascent RNA is synthesized. Our results also show that HIV-1 Tat protein stimulated DSIF and RNA pol II phosphorylation by P-TEFb during elongation. These findings reveal a molecular mechanism for the negative and positive regulation of transcriptional elongation at the HIV-1 promoter.

Cell Nucleus↗
PubMed2004

RNA polymerase II (Pol II)-TFIIF and Pol II-mediator complexes: the major stable Pol II complexes and their activity in transcription initiation and reinitiation.

Protein purification and depletion studies were used to determine the major stable forms of RNA polymerase II (Pol II) complexes found in Saccharomyces cerevisiae nuclear extracts. About 50% of Pol II is found associated with the general transcription factor TFIIF (Pol II-TFIIF), and about 20% of Pol II is associated with Mediator (Pol-Med). No Pol II-Med-TFIIF complex was observed. The activity of Pol II and the purified Pol II complexes in transcription initiation and reinitiation was investigated by supplementing extracts depleted of either total Pol II or total TFIIF with purified Pol II or the Pol II complexes. We found that all three forms of Pol II can complement Pol II-depleted extracts for transcription initiation, but Pol II-TFIIF has the highest specific activity. Similarly, Pol II-TFIIF has a much higher specific activity than TFIIF for complementation of TFIIF transcription activity. Although the Pol II-TFIIF and Pol II-Med complexes were stable when purified, we found these complexes were dynamic in extracts under transcription conditions, with a single polymerase capable of exchanging bound Mediator and TFIIF. Using a purified system to examine transcription reinitiation, we found that Pol II-TFIIF was active in promoting multiple rounds of transcription while Pol II-Med was nearly inactive. These results suggest that both the Pol II-Med and Pol II-TFIIF complexes can be recruited for transcription initiation but that only the Pol II-TFIIF complex is competent for transcription reinitiation.

Cell Extracts↗
PubMed2003

p53 functions through stress- and promoter-specific recruitment of transcription initiation components before and after DNA damage.

The tumor suppressor protein p53 regulates transcriptional programs that control the response to cellular stress. We show that distinct mechanisms exist to activate p53 target genes as revealed by marked differences in affinities and damage-specific recruitment of transcription initiation components. p53 functions in a temporal manner to regulate promoter activity both before and after stress. Before DNA damage, basal levels of p53 are required to assemble a poised RNA polymerase II initiation complex on the p21 promoter. RNA pol II is converted into an elongating form shortly after stress but before p53 stabilization. Proapoptotic promoters, such as Fas/APO1, have low levels of bound RNA pol II but undergo damage-induced activation through efficient reinitiation. Surprisingly, in a p53-dependent process key basal factors TAFII250 and TFIIB assemble into the transcription machinery in a stress- and promoter-specific manner, behaving as differential cofactors for p53 action after distinct types of DNA damage.

Apoptosis↗
PubMed2001

Reciprocity between O-GlcNAc and O-phosphate on the carboxyl terminal domain of RNA polymerase II.

The carboxyl terminal domain of RNA polymerase II has multiple essential roles in transcription initiation, promoter clearance, transcript elongation, and the recruitment of the RNA processing machinery. Specific phosphorylation events are associated with the spatial and temporal coordination of these different activities. The CTD is also modified by beta-O-linked GlcNAc on a subset of RNA Pol II molecules. Using synthetic CTD substrates, we show here that O-GlcNAc and phosphate modification of the CTD are mutually exclusive at the level of the enzymes responsible for their addition. In addition, we show that O-GlcNAc transferase and CTD kinase have different CTD repeat requirements for enzymatic activity. The Km values of the two enzymes for CTD substrates are in a similar range, indicating that neither enzyme has a distinct kinetic advantage. Thus, the in vivo regulation of O-GlcNAc and phosphate modification of the CTD may involve the differential association of these two enzymes with the CTD at specific stages during the transcription cycle. Furthermore, direct competition between OGT and CTD kinase in vivo could generate multiple functionally distinct isoforms of RNA Pol II. Taken together, these results suggest that O-GlcNAc may give rise to additional functional states of RNA Pol II and may coordinate with phosphorylation to regulate class II gene transcription.

Acetylglucosamine↗
PubMed1991

In vitro and in vivo analysis of the c-myc RNA polymerase III promoter.

The c-myc promoter has the unusual property of displaying both RNA polymerase II (Pol II) and RNA polymerase III (Pol III) activities. Both Pol II and Pol III utilize the same transcription initiation site. We have now examined the effects of mutations in crucial regions of the c-myc promoter to assess their effects on both transcriptional activities. In doing this we show that both Pol II and Pol III activities require sequences that are located within the stronger of the two principal c-myc promoter regions (P2). Further, we show that the Pol III activity using this initiation site does not require an A box or distal upstream sequences. Like the Pol II activity, it does require an intact TATA sequence and alterations at this site result in the simultaneous loss of both Pol II and Pol III activities. The superimposition of two apparently inseparable promoter activities makes it possible to consider common features, possible common protein elements in each holoenzyme complex, as well as a potential role for each enzyme in the regulated expression of the c-myc gene.

Base Sequence↗
PubMed2005

Computational analysis of plant RNA Pol-II promoters.

Plant promoters have not yet been thoroughly analyzed in terms of their structural and sequence dependent properties like curvature, periodicity and information content and our present study is an attempt in that direction. Results were compared with E. coli and yeast data to get some insight into the promoter organization. Promoters having the TATA box (TATA(+)) and those lacking the same (TATA(-)) were also analyzed separately. It was found that plant promoters have marked differences for all these properties when compared to E. coli and yeast. Bias for A+T was observed in promoters of all the three groups. Compared to E. coli and yeast, plant promoters showed intermediate values for A+T content as well as curvature. Analysis showed that curvature of core promoters is more pronounced than non-promoters. Information theoretic analysis of plant promoters reveal high information content at certain consensus regions such as -30 (TATA box) and +1 transcription start site (TSS); and have moderate values at other positions as well. This factor was taken into account while developing weight matrices. For certain threshold values, these weight matrices could pick up all true positives, and reduce false positives to a great extent in a test set. A new multi-parameterized prediction strategy has been proposed that uses a combination of sequence composition, curvature and position weight matrices for identification of plant promoters. This strategy was tested and validated with experimentally known promoter sequences. Our study is novel in using in silico approaches to study the sequence dependent properties of plant RNA Pol-II promoters and their prediction, and important as there is no dedicated promoter search tool for plants.

Base Sequence↗
PubMed1997

Analysis of a cAMP-responsive activator reveals a two-component mechanism for transcriptional induction via signal-dependent factors.

We have examined the mechanism by which the cAMP-responsive factor CREB stimulates target gene expression following its phosphorylation at Ser-133. Using an in vitro transcription assay, we found that two signals were required for target gene activation: a phospho(Ser-133)-dependent interaction of CREB with RNA polymerase II via the coactivator CBP and a glutamine-rich domain interaction with TFIID via hTAF(II)130. The adenovirus E1A oncoprotein was found to inhibit phospho(Ser-133) CREB activity by binding to CBP and specifically blocking recruitment of RNA Pol II to the promoter. Our results suggest that the recruitment of CBP-RNA Pol II complexes per se is not sufficient for transcriptional activation and that activator-mediated recruitment of TFIID is additionally required for induction of signal-dependent genes.

Adenovirus E1A Proteins↗
PubMed2002

Localization of the yeast RNA polymerase I-specific subunits.

The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling. A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14. The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II. Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA. The location of the RNA pol I-specific subunits is correlated with their biological activity.

Cryoelectron Microscopy↗
PubMed1999

RNA polymerase II targets pre-mRNA splicing factors to transcription sites in vivo.

Biochemical evidence indicates that pre-mRNA splicing factors physically interact with the C-terminal domain of the largest subunit of RNA polymerase II. We have investigated the in vivo function of this interaction. In mammalian cells, truncation of the CTD of RNA pol II LS prevents the targeting of the splicing machinery to a transcription site. In the absence of the CTD, pre-mRNA splicing is severely reduced. The presence of unspliced RNA alone is not sufficient for the accumulation of splicing factors at the transcription site, nor for its efficient splicing. Our results demonstrate a critical role for the CTD of RNA pol II LS in the intranuclear targeting of splicing factors to transcription sites in vivo.

Amanitins↗
PubMed2005

Mechanism of site-specific psoralen photoadducts formation in triplex DNA directed by psoralen-conjugated oligonucleotides.

Triplex-formation oligonucleotides attached with a photoreactive psoralen molecule (psoTFO) can be used to induce site-specific DNA damage and to control gene expression. Inhibition of transcription by psoralen-cross-linked triplexes results in both arrest and termination of RNA Pol II transcriptional complexes during elongation. To understand the relationship between triplex psoralen cross-linking products and the fate of RNA Pol II elongation complexes, it is important to delineate the mechanism for creating site-specific psoralen photoadducts in a target duplex DNA. To investigate the mechanism of photoadduct-formation by psoralen photo-cross-linking, triplex structures were generated by targeting a DNA duplex with psoTFOs of different lengths. The psoralen photoadducts were then analyzed after UV irradiation, which initiates the psoralen cross-linking reaction. Our results demonstrated that UV irradiation of triplexes formed between a psoTFO and a DNA duplex generated two distinct groups of psoralen photoadducts: monoadducts and psoralen interstrand cross-link products. The formation of these psoralen photoadducts was also photoreversible through exposure to short wavelength UV irradiation. The length of a psoTFO was shown to establish the position at which psoralen was added to the target DNA duplex and determined which photoadducts products formed predominantly. Kinetic experiments that monitored the formation of the psoralen photoadducts also suggested that the length of the psoTFO influenced which photoadducts were preferentially formed at faster rates. Taken together, these studies provide new insight into the mechanism associated with the formation of psoralen photoadducts that are directed by psoTFO during triplex formation.

Base Sequence↗
PubMed2002

Inhibition of HTLV-1 transcription by cyclin dependent kinase inhibitors.

HTLV-1 is the etiologic agent for adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), where viral replication and transformation are largely dependent upon modification of regulatory and host cell cycle proteins. The mechanism of HTLV-1 transformation appears to be distinct from that of many known chronic or acute leukemia viruses and is related to the viral activator Tax. Here we show that cyclin E, can associate tightly with the coactivator p300 and Pol II complex in HTLV-1 infected cells. The cyclin E associated complex is kinase active and phosphorylates the carboxy terminal domain of RNA Pol II. More importantly, p21/Waf1, a well-known cdk inhibitor at the G1/S border, inhibits transcription of HTLV-1 in both transfections and in in vitro transcription assays. Finally, specific cdk chemical inhibitors, functionally similar to cellular cdkIs, such as p21/Waf1 which inhibits cyclin E/cdk2 activity, also inhibit transcription of the HTLV-1 promoter. In particular, Purvalanol A, with an IC50 of 0.035 microm inhibits activated, but not basal transcription, as well as HTLV-1 infected cells. Collectively, the role of cyclin E/cdk2 in HTLV-1 infected cells and its involvement in RNA Pol II phosphorylation is discussed.

Amino Acid Sequence↗
PubMed2005

Expression-based discovery of variation in the human glutathione S-transferase M3 promoter and functional analysis in a glioma cell line using allele-specific chromatin immunoprecipitation.

Discovery and functional evaluation of biologically significant regulatory single nucleotide polymorphisms (SNP) in carcinogen metabolism genes is a difficult challenge because the phenotypic consequences may be both transient and subtle. We have used a gene expression screening approach to identify a functional regulatory SNP in glutathione S-transferase M3 (GSTM3). Anttila et al. proposed that variation in GSTM3 expression was affected by exposure to cigarette smoke and inheritance of the GSTM1-null genotype. To investigate the mechanism of GSTM3 expression was affected by exposure to cigarette smoke and inheritance of the GSTM1-null genotype. To investigate the mechanism of GSTM3 expression variation, we measured GSTM3 expression in lymphoblast cells from a human Centre d'Etude du Polymorphisme Humain family and observed a low expression phenotype. Promoter sequencing revealed two novel GSTM3 promoter SNPs: A/C and A/G SNPs, 63 and 783 bp upstream of the codon 1 start site, respectively. In this pedigree, the two children homozygous for the -63C/C genotype had 8-fold lower GSTM3 expression relative to the two children with the -63A/A genotype, with no association between A-783G SNP and GSTM3 expression. Further evaluation using genotyped glioma cell lines and with luciferase reporter constructs showed that the -63C allele was associated with lower GSTM3 expression (P < 0.0001 and P < 0.003). RNA pol II chromatin immunoprecipitation was combined with quantitative probed-based allelic discrimination genotyping to provide direct evidence of a 9-fold reduced RNA pol II binding capacity for the -63C allele. These results show that the GSTM3 -63C allele strongly affects gene expression in human cell lines and suggests that individuals who carry the low expression allele may be deficient in glutathione transferase catalyzed biological functions.

Base Sequence↗
PubMed2026

Chromatin context shapes SPT5 regulation of promoter-proximal Pol II, fine-tuning gene expression changes during Drosophila embryogenesis.

Transcription involves initiation, pausing, elongation, and termination. Suppressor of Ty5 (SPT5) regulates promoter-proximal pausing and elongation, but how it orchestrates both steps during dynamic developmental changes in gene expression remains unclear. Here, using rapid optogenetic depletion in Drosophila embryos, we uncover different consequences of SPT5 removal at different developmental stages. In early embryos, SPT5 depletion causes a shift of RNA polymerase II (Pol II) from the canonical pausing site to the +1 nucleosome, which is strongly positioned. In late embryos, SPT5 depletion similarly reduces pausing at the canonical site, but the transcriptional machinery can overcome the +1 nucleosome-which appears more labile at this time point-moving into the gene body. This results in lethality and both up- and downregulation of expression, depending on the balance between Pol II entering the gene body and defective elongation. This is intensified for genes naturally increasing or decreasing their expression, indicating that SPT5 contributes to fine-tuning dynamic expression changes.

+1 nucleosome↗
PubMed1997

Characterization of antinucleolar antibody reactivity in patients with systemic sclerosis and their relatives.

OBJECTIVE: To study the prevalence and specificity of antinucleolar antibodies (ANoA) in patients with systemic sclerosis (SSc), their spouses, and their first-degree relatives, and to investigate whether SSc family members have greater frequency of ANoA than expected. METHODS: The sera of 58 SSc probands, 4 first-degree relatives with SSc, 215 first-degree relatives without SSc, and 24 spouses were screened for ANoA by indirect immunofluorescence (IF), and nucleolar antigens were characterized by immunoprecipitation (IP) of 35S methionine labeled K562 cell extracts. Sera from 118 randomly chosen family members without SSc were separately compared with 120 age and sex matched blood donor controls. RESULTS: Antinucleolar reactivity was detected by IF in 25 patients with SSc (40.3%), in 33 non-SSc relatives (15.3%), and in 4 spouses (16.7%). Twenty-four sera had autoantibodies to defined nucleolar antigens by IP (seven Pm-Scl, ten RNA polymerase (pol) I, four U3 RNP, three Th RNP), and all were from patients with SSc (38.7%). No serum had more than one type of nucleolar-specific autoantibody. Four sera had autoantibodies to topoisomerase I (topo I) and RNA pol II, one of which also recognized RNA pol I and RNA pol III. Antinucleolar IF was significantly more common in the unaffected first-degree relatives of patients with SSc (18.1%) than in controls (8.3%; p < 0.05). A small number of sera from both relatives and controls recognized bands by IP, none of which was identified as a SSc-specific autoantigen. CONCLUSION: Although antinucleolar reactivity is more common in the first-degree relatives of patients with SSc than controls, SSc associated ANoA are only present in patients with the disease, and appear to be mutually exclusive.

Antibodies, Antinuclear↗