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PubMed1976

Template properties of human DNA-dependent RNA polymerase II.

DNA-dependent RNA polymerase II has been purified from lymphocytes of patients with chronic lymphocytic leukemia (CLL). Form II polymerase and Escherichia coli RNA polymerase have been used to study the transcription of human DNA and chromatin. The hybridization kinetics of the transcripts of DNA and chromatin by the human polymerase are quite different; chromatin transcripts hybridized to DNA at a much slower rate than DNA transcripts, whereas the transcripts of DNA and chromatin by bacterial polymerase have similar hybridization kinetics.

Centrifugation, Density Gradient↗

PubMed1987

The 180 KDa polypeptide contains the DNA-binding domain of RNA polymerase II.

Purification of RNA polymerase II from chicken myeloblastosis (leukemia) cells to homogeneity and subsequent structural analysis of the purified enzyme revealed that the enzyme contained seven polypeptides with molecular masses ranging from 27 KDa to 220 KDa. Inclusion of protease inhibitors in the buffer system during purification significantly increased the molar ratio of the largest (220 KDa) polypeptide to the second largest (180 KDa) polypeptide. However, proteolytic conversion of the 220 KDa to 180 KDa polypeptide did not inhibit the DNA binding activity of the enzyme. The enzyme, after dissociation into subunits in a SDS-polyacrylamide gel containing urea was blotted onto a nitrocellulose filter. The filter was incubated with 32P-labeled calf thymus DNA and both the 220 KDa and 180 KDa polypeptides of the enzyme bind DNA, suggesting that the DNA-binding site of the enzyme resides on the 180 KDa polypeptide of the largest subunit.

Animals↗

PubMed1997

Identification of the region in yeast S-II that defines species specificity in its interaction with RNA polymerase II.

Yeast S-II was found to stimulate yeast RNA polymerase II only and not mouse RNA polymerase II. To identify the molecular region of S-II that defines species specificity, we constructed six hybrid S-II molecules consisting of three regions from yeast and/or Ehrlich cell S-II and examined their activity in terms of RNA polymerase II specificity and suppression of 6-azauracil sensitivity in the yeast S-II null mutant. We found that the region 132-270 (amino acid positions) of yeast S-II is indispensable for specific interaction with yeast RNA polymerase II in vitro and for suppression of 6-azauracil sensitivity in vivo. The corresponding region of Ehrlich cell S-II, the region 132-262, was also shown to be essential for its interaction with mouse RNA polymerase II. This region is known to be less conserved than the N- and C-terminal regions in the S-II family suggesting that it is important in the interaction with transcription machinery proteins in a tissue and/or species-specific manner.

Amino Acid Sequence↗

PubMed2006

Molecular architecture and conformational flexibility of human RNA polymerase II.

Transcription by RNA polymerase II (RNAPII) is a central process in eukaryotic gene regulation. While atomic details exist for the yeast RNAPII, characterization of the human complex lags behind, mostly due to the inability to obtain large quantities of purified material. Although the complexes have the same protein composition and high sequence similarity, understanding of transcription and of transcription-coupled DNA repair (TCR) in humans will require the use of human proteins in structural studies. We have used cryo-electron microscopy, image reconstruction, and variance analysis to characterize the structure and dynamics of human RNAPII (hRNAPII). Our studies show that hRNAPII in solution parallels the conformational flexibility of the yeast structures crystallized in different states but also illustrate a more extensive conformational range with potential biological significance. This hRNAPII study will serve as a structural platform to build up higher-order transcription and TCR complexes and to gain information that may be unique to the human RNAPII system.

Cryoelectron Microscopy↗

PubMed1988

Transcription of left-handed Z-DNA templates: increased rate of single-step addition reactions catalyzed by wheat germ RNA polymerase II.

Wheat germ RNA polymerase II is able to transcribe polynucleotide templates in the poly-[d(G-C)] family, adopting either the right-handed B or left-handed Z conformations depending on the ionic environment and temperature. Thus, with poly[d(G-C)] either the B state (in MgCl2) or the associated Z* state (in MnCl2) can be established. Poly[d(G-m5C)] adopts the Z form readily in MgCl2, and poly-[d(G-br5C)] can be regarded as being "constitutively" in the Z state. In transcription studies with CpG as a primer and templates in the left-handed conformation, it is found that the rate of productive elongation, i.e., the synthesis of poly[r(G-C)], is depressed, in accordance with the results of previous studies. However, with a single triphosphate substrate, CTP, the rate of formation of the first phosphodiester bond, i.e., the synthesis of CpGpC, is about 4-fold greater with both the Z and Z* templates than with B-DNA. This transcriptional activity is also catalytic in the sense that product concentrations exceed that of the enzyme. The synthesis of CpGpC is reduced in the presence of GTP. However, the apparent Km value for GTP utilization is lower for the trinucleotide synthesis (0.1 microM) than that obtained for productive elongation (0.8 microM), a result that also holds for B-DNA templates. All transcription reactions are specifically inhibited by the fungal toxin alpha-amanitin, and, in the case of the left-handed templates, by monoclonal anti-Z-DNA antibodies. The relative probabilities of single-step addition and productive elongation imply that the major distinction between transcription of templates in the B and Z conformations involves a step following the synthesis of the first phosphodiester bond. As a result, fully competent elongation complexes do not form on the left-handed DNA.

Chlorides↗

PubMed1995

RNA polymerase III dependence of the human L1 promoter and possible participation of the RNA polymerase II factor YY1 in the RNA polymerase III transcription system.

From the general views of the eukaryotic transcription systems, L1 (or L1-like) retrotransposons that encode some proteins are unusual. L1, unlike other protein-coding elements, is transcribed through an internal promoter. And the L1 internal promoter, unlike other internal promoters, is thought to be RNA polymerase II (pol II) dependent, because the L1 transcript has a large size (approximately 6 kb), protein coding capacity and a 3' terminal polyadenylation signal followed by a poly(A) tail, and also because transcription from the promoter of Drosophila L1-like element jockey was highly sensitive to alpha-amanitin. However, our in vitro transcription study reveals that transcription from the human L1 promoter is highly sensitive to tagetitoxin, a selective inhibitor of RNA polymerase III (pol III), but insensitive to 1 micrograms/ml of alpha-amanitin, indicating that the human L1 promoter is pol III-dependent. The pol III dependence is further supported by our observation that L1 and pol III-dependent tRNA gene promoters share a common nuclear factor YY1. There is evidence that YY1 is also a pol II transcription factor. We thus propose that YY1 is a possible member of the pol III transcription system.

Base Sequence↗

PubMed1996

Association between 36- and 13.6-kDa alpha-like subunits of Arabidopsis thaliana RNA polymerase II.

Two subunits in RNA polymerase II (e.g. RPB3 and RPB11 in yeast) and two subunits common to RNA polymerases I and III (e.g. AC40 and AC19 in yeast) contain one or two motifs related to the alpha subunit in prokaryotic RNA polymerases. We have sequenced two different cDNAs (AtRPB36a and AtRPB36b), the two corresponding genes from Arabidopsis thaliana that are homologs of yeast RPB3, and an Arabidopsis cDNA (AtRPB13.6) that is a homolog of yeast RPB11. The B36a subunit is the predominant B36 subunit associated with RNA polymerase II purified from Arabidopsis suspension culture cells, and this subunit has a stoichiometry of about 1. Results from protein association assays showed that the B36a and B36b subunits did not associate, but each of these subunits did associate with the B13.6 subunit in vivo and in vitro. Two motifs in the B36b subunit related to the prokaryotic alpha subunit were shown to be required for the in vitro interactions with the B13.6 subunit. Our results suggest that the B36 and B13.6 subunits associate to form heterodimers in Arabidopsis RNA polymerase II like the AC40 and AC19 heterodimers reported for yeast RNA polymerases I and III but unlike the B44 homodimers reported for yeast RNA polymerase II.

Amino Acid Sequence↗

PubMed1995

General requirement for RNA polymerase II holoenzymes in vivo.

Yeast RNA polymerase II holoenzymes have been described that consist of RNA polymerase II, a subset of general transcription factors, and nine SRB regulatory proteins. The feature that distinguishes the RNA polymerase II holoenzymes from other forms of RNA polymerase II in the cell is their tight association with SRB proteins. We investigated the fraction of genes that require SRB proteins in vivo by examining the effect of temperature-sensitive mutations in SRB genes on transcription by RNA polymerase II. Upon transfer to the restrictive temperature, there is a rapid and general shutdown of mRNA synthesis in srb mutant cells. These data, combined with the observation that essentially all of the SRB protein in cells is tightly associated with RNA polymerase II molecules, argue that SRB-containing holoenzymes are the form of RNA polymerase II recruited to most promoters in the cell.

Base Sequence↗

PubMed1994

Transcriptional activation of NFI/CTF1 depends on a sequence motif strongly related to the carboxyterminal domain of RNA polymerase II.

Initiation of RNA polymerase II-directed transcription is mediated by DNA sequence specific activator proteins interacting with components of the basal transcription machinery. NFI/CTF is a family of such binding proteins which have been shown to stimulate transcription via proline-rich activation domains. In order to identify residues crucial for its activator function, a pool of CTF1 mutants was cloned and fused to the bacterial repressor LexA. Transcriptional activation of these constructs was monitored in a Saccharomyces cerevisiae reporter assay. Our studies reveal the existence of a core domain in CTF1 between residues 463 and 508 essential for transcriptional activation functions. It contains the sequence motif SPTSPSYSP, which is strongly related to the heptapeptide repeat YSPTSPS present in the carboxyterminal domain (CTD) of RNA polymerase II. Removal of the entire CTD related motif, as well as substitution of key amino acids therein, abolish CTF1 mediated transcriptional activation.

Amino Acid Sequence↗

PubMed1996

Human immunodeficiency virus type 1 and 2 Tat proteins specifically interact with RNA polymerase II.

The Tat-responsive region (TAR) element is a critical RNA regulatory element in the human immunodeficiency virus (HIV) long terminal repeat, which is required for activation of gene expression by the transactivator protein Tat. Recently, we demonstrated by gel-retardation analysis that RNA polymerase II binds to TAR RNA and that Tat prevents this binding even when Tat does not bind to TAR RNA. These results suggested that direct interactions between Tat and RNA polymerase II may prevent RNA polymerase II pausing and lead to Tat-mediated increases in transcriptional elongation. To test this possibility, we performed protein interaction studies with RNA polymerase II and both the HIV-1 and the closely related HIV-2 Tat protein. These studies indicated that both the HIV-1 and HIV-2 Tat proteins could specifically interact with RNA polymerase II. Mutagenesis of both HIV-1 and HIV-2 Tat demonstrated that the basic domains of both the HIV-1 and HIV-2 Tat proteins were required for this interaction. Furthermore, "far Western" analysis suggested that the largest subunit of RNA polymerase II was the site for interaction with Tat. The interactions between Tat and RNA polymerase II were of similar magnitude to those detected between RNA polymerase II and the cellular transcription factor RAP30, which stably associates with RNA polymerase II during transcriptional elongation. These studies are consistent with the model that RNA polymerase II is a cellular target for Tat resulting in Tat-mediated increases in transcriptional elongation from the HIV long terminal repeat.

Animals↗

PubMed1988

Formation of transcription preinitiation complexes with an amanitin-resistant RNA polymerase II.

Accurate transcription by RNA polymerase II has been shown to require multiple factors which participate in a number of intermediate steps prior to initiation. The last detectable step before initiation is the formation of an activated or rapid start complex which is template-associated. In this study we used two phenotypically distinguishable forms of mammalian RNA polymerase II to examine the requirement for specific factors in the formation of the activated complex. RNA polymerase II was purified from a mutant cell line which was resistant to levels of alpha-amanitin that are toxic for normal cells. When added to a polymerase-dependent transcription system consisting of HeLa factors and adenovirus DNA, the mutant polymerase accurately transcribed the template in an amanitin-resistant fashion. This amanitin-resistant transcription was competitively inhibited when wild-type polymerase was also added to the system. Preincubation of amanitin-resistant polymerase with DNA and factors produced a polymerase-associated complex, defined by its resistance to exchange for the amanitin-sensitive polymerase. Complex formation required the presence of polymerase, DNA, and the HeLa factors during the preincubation but did not require the presence of nucleotides. Complexes were template-associated, as shown by their inability to exchange onto a second template. Thus, prior to initiation, RNA polymerase II forms a stable association with the DNA template in an activated complex.

Amanitins↗

PubMed1976

Purification of a factor from Ehrlich ascites tumor cells specifically stimulating RNA polymerase II.

A factor stimulating RNA polymerase II from Ehrlich ascites tumor cells was purified. The final preparation appeared almost homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 38 000. The endonuclease activity of about 10 mug of purified factor, if any was well below the 10(-5) mug equivalent of pancreatic deoxyribonuclease, indicating that the stimulation of RNA synthesis by this factor was not due to contaminating endonuclease. This factor specifically stimulated RNA polymerase II on native DNA as template and did not affect RNA polymerase I at all. The molecular size of RNA synthesized in the presence of this factor increased markedly compared with that synthetized by RNA polymerase II alone.

Animals↗

PubMed1990

A suppressor of an RNA polymerase II mutation of Saccharomyces cerevisiae encodes a subunit common to RNA polymerases I, II, and III.

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.

Base Sequence↗

PubMed2005

RNA-interference-directed chromatin modification coupled to RNA polymerase II transcription.

RNA interference (RNAi) acts on long double-stranded RNAs (dsRNAs) in a variety of eukaryotes to generate small interfering RNAs that target homologous messenger RNA, resulting in their destruction. This process is widely used to 'knock-down' the expression of genes of interest to explore phenotypes. In plants, fission yeast, ciliates, flies and mammalian cells, short interfering RNAs (siRNAs) also induce DNA or chromatin modifications at the homologous genomic locus, which can result in transcriptional silencing or sequence elimination. siRNAs may direct DNA or chromatin modification by siRNA-DNA interactions at the homologous locus. Alternatively, they may act by interactions between siRNA and nascent transcript. Here we show that in fission yeast (Schizosaccharomyces pombe), chromatin modifications are only directed by RNAi if the homologous DNA sequences are transcribed. Furthermore, transcription by exogenous T7 polymerase is not sufficient. Ago1, a component of the RNAi effector RISC/RITS complex, associates with target transcripts and RNA polymerase II. Truncation of the regulatory carboxy-terminal domain (CTD) of RNA pol II disrupts transcriptional silencing, indicating that, like other RNA processing events, RNAi-directed chromatin modification is coupled to transcription.

Argonaute Proteins↗

PubMed1981

Stimulation of messenger ribonucleic acid synthesis in isolated nuclei by a protein that stimulates RNA polymerase II.

A factor stimulating RNA polymerase II purified from Ehrlich ascites tumor cells was found to stimulate alpha-amanitin-sensitive RNA synthesis in nuclei isolated from spleen cells of anemic mice, though less than it stimulated purified RNA polymerase II. The fidelity of the resulting RNA synthesis was monitored by measuring the stimulation of globin mRNA synthesis. Globin mRNA was measured quantitatively by DNA-RNA hybridization by using plasmid DNA containing globin DNA sequences. Results showed that the synthesis of globin mRNA was enhanced in isolated nuclei in the presence of this factor coinciding with an increase of overall alpha-amanitin-sensitive RNA synthesis. Thus, it was concluded that an externally added factor did not stimulate random transcription but meaningful RNA synthesis in isolated nuclei.

Anemia↗

PubMed1977

Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II.

DNA-dependent RNA polymerase II from calf thymus has been successfully purified using polythylenimine precipitation. Thus, 5-6 mg of nearly homogeneous homogeneous trna polymerase II (greater than 96% pure) can be prepared from 1 kg of calf thymus with three chromatography steps following extraction and precipitation of the enzyme from the polyethylenimine pellet. This procedure eliminates the high salt extraction of chromatin previously used in purification of this enzyme and makes possible the large scale preparation of mammalian RNA polymerase II. Calf thymus polymerase II prepared by this method is greater than 90% form IIb and consists of ten different subunits having the following molecular weights: 180 000; 145 000; 36 000; 25 000; 20 000; 18 500; 16 000; 15 000; 12 000; 11 500. The homologous enzyme isolated from wheat germ is greater than 90% form IIa and contains subunits of the following molecular weights: 206 000; 145 000; 44 000-47 000; 24 500; 21 000; 19 000; 17 000; 14 000; 13 500. The wheat germ and calf thymus enzymes exhibit similar subunits structures, but the molecular weights of individual subunits are clearly different between the enzymes. Wheat germ RNA polymerase II is 50% inhibited by 0.271 microng/mL of alpha-amanitin, a level 30-fold higher than that found for calf thymus RNA polymerase II. These enzymes are further distinguished by the absence of antigenic cross reactivity.

Animals↗

PubMed1981

In vitro transcription by wheat germ RNA polymerase II. Initiation of RNA synthesis on relaxed, closed circular template.

We have developed a novel method for studying the ability to wheat germ RNA polymerase II to initiate transcription in vitro on unnicked duplex DNA template. Our template is a plasmid which has been relaxed in vitro with wheat germ DNA topoisomerase I. The plasmid consists of SV40 cloned in pBR322. We transcribe briefly to synthesize highly labeled nascent RNA, then fractionate the resulting ternary complexes on an agarose gel to separate relaxed, closed circular DNA from other DNA species contaminating the template preparation. Under optimal conditions, we recover about half of the total RNA synthesized in complex with relaxed, closed circular DNA. The remaining RNA is in association with other DNA species or is free in solution. The principal conclusions we draw from these studies are: 1) relaxed, closed circular DNA can serve as a template for in vitro transcription, although the relative efficiencies of transcription are such that even a few per cent contamination with nicked or supercoiled template can cause transcription from these forms to dominate the reaction. 2) Initiation occurs on approximately 1 in 25 cloned circular molecules in the course of a 5-min reaction, under optimal conditions. This transcription is not predominantly from SV40 in vivo promoters and is either random or from a sufficient number of sites as to appear random. 3) There is a striking dependence of initiation frequency on purine nucleoside triphosphate concentration, especially with GTP.

DNA Topoisomerases, Type I↗

PubMed1992

Mechanism of assembly of the RNA polymerase II preinitiation complex. Evidence for a functional interaction between the carboxyl-terminal domain of the largest subunit of RNA polymerase II and a high molecular mass form of the TATA factor.

Genetic evidence argues that the highly conserved carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II functions directly in the regulation of transcription of many eukaryotic genes. The observation that partial deletion of the CTD of yeast RNA polymerase II reduces the ability of the enzyme to respond to signals from a variety of upstream activating sequences led to the proposal that the CTD plays a role in the dialogue between regulatory factors that bind upstream activating sequences and the "general" or "basal" transcription factors associated with RNA polymerase II at the promoter (Scafe, C., Chao, D., Lopes, J., Hirsch, J. P., Henry, S., and Young, R. A. (1990) Nature 347, 491-494). Biochemical evidence for an interaction of the CTD with specific components of the basal transcription apparatus, however, has been lacking. To identify target(s) for CTD action, we probed steps in assembly of the RNA polymerase II preinitiation complex with monoclonal antibodies specific for the CTD. Our findings reveal a novel interaction of the CTD with a high molecular mass form of the TATA factor. This interaction occurs during binding of RNA polymerase II to its promoter and requires the action of additional basal transcription factors; it is not observed when the single-subunit yeast transcription factor IID serves as the TATA factor.

Animals↗