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PubMed1992

Purification and properties of Saccharomyces cerevisiae RNA polymerase II general initiation factor a.

RNA polymerase II initiation factor a was purified to apparent homogeneity from yeast whole cell extract and consisted of two highly charged polypeptides with apparent masses of 66 and 43 kDa. Separation and renaturation of the subunits showed that both were required for transcription activity. The native mass of factor a was estimated to be 240-260 kDa by gel filtration, but its sedimentation rate in a glycerol gradient was similar to that of a much smaller globular protein, suggesting an extended conformation. Factor a was required for utilization of six different eukaryotic promoters in vitro, indicating a general role in promoter-directed transcription by yeast RNA polymerase II.

Centrifugation, Density Gradient↗
PubMed1997

In vitro transcription of human T-cell leukemia virus type 1 is RNA polymerase II dependent.

The HTLV-1 promoter directs RNA polymerase II transcription of viral genomic RNA in vivo. However, it has been reported that in vitro, a unique RNA polymerase, with characteristics of RNA polymerases II and III, is capable of HTLV-1 transcription (G. Piras, F. Kashanchi, M. F. Radonovich, J. F. Duvall, and J. N. Brady, J. Virol. 68:6170-6179, 1994). To further characterize the polymerase involved in HTLV-1 transcription in vitro, runoff transcription assays were performed with a variety of extracts and RNA polymerase inhibitors. Under all in vitro reaction conditions tested, RNA polymerase II appeared to be the only polymerase capable of correct transcriptional initiation from the HTLV-1 promoter. Synthesis of the specific HTLV-1 RNA transcript showed sensitivities to the RNA polymerase inhibitors tagetitoxin and alpha-amanitin that are consistent with RNA polymerase II transcription. Together, these data indicate that in vitro, as in vivo, the HTLV-1 promoter directs transcription by RNA polymerase II.

Amanitins↗
PubMed1983

Structural relationship between the large subunits of calf thymus RNA polymerase II.

Purified calf thymus RNA polymerase II is composed primarily of species IIA and IIB. These enzymes differ in the apparent molecular weight of their largest subunit, designated IIa and IIb for enzyme forms IIA and IIB, respectively. Both enzyme forms contain an additional high molecular weight subunit designated IIc. The structural relationship between subunits IIa, IIb, and IIc, labeled with 125I under both native and denaturing conditions, has been analyzed by two-dimensional peptide mapping. Native RNA polymerase II was iodinated and subunits IIa, IIb, and IIc purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The subunits were then digested with either trypsin or thermolysin and the 125I-labeled peptides resolved by thin layer electrophoresis in the first dimension and chromatography in the second dimension. Similar peptide maps were obtained for each of the three large subunits, suggesting that subunits IIa, IIb, and IIc are related in primary sequence. Alternatively, RNA polymerase subunits IIa, IIb, and IIc were purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, eluted from the gel, and then iodinated. The use of denatured subunits as substrate for the iodination eliminates the differential reactivity of specific tyrosine residues imposed by the structure of the native protein. Under these labeling conditions, the tryptic and thermolytic peptide maps of subunits IIa and IIb are nearly identical but bear much less resemblance to the peptide maps of subunit IIc than with the previous labeling procedure. These results suggest that subunits IIa and IIb are closely related in primary sequence but cannot establish whether these subunits are the products of closely related genes or are related by processing at the level of primary transcript or primary translation product. Subunit IIc bears a more distant relationship to subunits IIa and IIb. Possible reasons why this homology is only apparent in peptide maps from subunits labeled in the native enzyme are discussed.

Animals↗
PubMed2001

RNA polymerase II and III transcription factors can stimulate DNA replication by modifying origin chromatin structures.

Many transcription factors are multifunctional and also influence DNA replication. So far, their mechanism of action has remained elusive. Here we show that a DNA-binding protein could rely on the same biochemical activity that activates transcription to stimulate replication from the yeast chromosomal ARS1 origin. Unexpectedly, the ability to stimulate replication from this origin was not restricted to polymerase II transcription factors, but was a property shared by polymerase III factors. Furthermore, activation of replication did not depend on the process of transcription, but rather on the ability of DNA-binding transcription factors to remodel chromatin. The natural ARS1 activator Abf1 and the other transcription factors that stimulated replication remodeled chromatin in a very similar manner. Moreover, the presence of a histone H3 mutant that was previously shown to generally increase transcription also facilitated replication from ARS1 and partially compensated for the absence of a transcription factor. We propose that multifunctional transcription factors work by influencing the chromatin architecture at replication origins so as to generate a structure that is favorable to the initiation of replication.

Binding Sites↗
PubMed1990

[Highly selective affinity labeling of DNA-dependent RNA-polymerase II from human placenta].

RNA polymerase II from human placenta was affinity labelled in crude preparation using two-step technique, which includes treatment of the enzyme with an aldehyde-containing reactive analogue of ATP, ADP or AMP in the presence of poly[d(A-T)] followed (after borohydride reduction) by the elongation of the attached label with [alpha-32P]UTP. A polypeptide of the molecular mass ca. 140 kDa proved to be the labelling target. No labelling was observed in the absence of poly[d(A-T)] or the reagent or in the presence of alpha-amanitin. All the results suggest the attachment of the affinity reagents to the second-largest subunit of the human RNA polymerase II, which therefore takes part in the initiation substrate's binding.

Affinity Labels↗
PubMed1994

Affinity labeling and measurement of DNA-induced conformation change in RNA polymerase II.

The inhibition of RNA polymerase II by 8-azido-2'-O-(1-naphthalenesulfonyl)- ATP (AN-ATP) was found to be reversible in the dark and competitive with ATP, with KI = 2.2 +/- 0.2 microM and KATP = 20 +/- 0.5 microM, but noncompetitive with CTP and GTP, with dissociation constants KCTP = 4.4 +/- 0.4 microM and KGTP = 19.4 +/- 0.7 microM. Under UV irradiation the enzyme was irreversibly labeled by AN-[alpha-32P]ATP. A linear plot of the relative specific activities of labeled enzyme samples after gel filtration vs the number of labels per enzyme molecule shows that each covalent label completely inactivates an enzyme molecule. Therefore the labeling has maximal specificity at an essential specific ATP site which is not the substrate-binding site for the polymerization reaction. The fluorescent AN-ATP-labeled enzyme, with absorbance maximum at 310 nm and emission maximum at 370 nm, can still bind noncovalently a second ligand, 2'-O-dansyl-ATP, with absorbance maximum at 365 nm and emission maximum at 560 nm, at the substrate-binding site. Measurement of energy transfer between the two fluorescent labels gave R = 23.8 +/- 0.4 A as the average distance between them and Kd = 31 +/- 0.1 microM for the bound 2'-O-dansyl-ATP in the absence of DNA template. The addition of either thymus DNA or poly[d(A-T)] to the system changed these values to R = 25.6 +/- 0.4 A and Kd = 53 +/- 0.4 microM. These results indicate that the binding of RNA polymerase II to DNA template triggers a conformation change in the enzyme molecule.

Adenosine Triphosphate↗
PubMed1998

BRCA1 protein is linked to the RNA polymerase II holoenzyme complex via RNA helicase A.

The breast cancer specific tumour suppressor protein, BRCA1 (refs 1,2), activates transcription when linked with a DNA-binding domain and is a component of the RNA polymerase II (Pol II) holoenzyme. We show here that RNA helicase A (RHA) protein links BRCA1 to the holoenzyme complex. The region of BRCA1 which interacts with RHA and, thus, the holoenzyme complex, corresponds to subregions of the BRCT domain of BRCA1 (ref. 9). This interaction was shown to occur in yeast nuclei, and expression in human cells of a truncated RHA molecule which retains binding to BRCA1 inhibited transcriptional activation mediated by the BRCA1 carboxy terminus. These data are the first to identify a specific protein interaction with the BRCA1 C-terminal domain and are consistent with the model that BRCA1 functions as a transcriptional coactivator.

Animals↗
PubMed1993

Identification of autoantibodies to RNA polymerase II. Occurrence in systemic sclerosis and association with autoantibodies to RNA polymerases I and III.

In this study, autoantibodies to RNA polymerase II from sera of patients with systemic sclerosis have been identified and characterized. These antibodies immunoprecipitated polypeptides of 220 kD (IIA) and 145 kD (IIC), the two largest subunits of RNA polymerase II, and bound both subunits in immunoblots. These polypeptides were immunoprecipitated by the anti-RNA polymerase II monoclonal antibody 8WG16, which recognizes the carboxyl-terminal domain of the 220-kD subunit, and their identity to the proteins bound by human sera was confirmed in immunodepletion studies. Sera with anti-RNA polymerase II antibodies also immunoprecipitated proteins that were consistent with components of RNA polymerases I and III. In vitro transcription experiments showed that the human antibodies were an effective inhibitor of RNA polymerase II activity. In indirect immunofluorescence studies, anti-RNA polymerase II autoantibodies stained the nucleoplasm, as expected from the known location of RNA polymerase II, and colocalized with the anti-RNA polymerase II monoclonal antibody. The human sera also stained the nucleolus, the location of RNA polymerase I. From a clinical perspective, these antibodies were found in 13 of 278 patients with systemic sclerosis, including 10 with diffuse and three with limited cutaneous disease, but were not detected in sera from patients with other connective tissue diseases and from normal controls. We conclude that anti-RNA polymerase II antibodies are specific to patients with systemic sclerosis, and that they are apparently associated with antibodies to RNA polymerases I and III. These autoantibodies may be useful diagnostically and as a probe for further studies of the biological function of RNA polymerases.

Adenoviridae↗
PubMed2006

Biochemical characterization of Trypanosoma brucei RNA polymerase II.

In Trypanosoma brucei, transcription by RNA polymerase II accounts for the expression of the spliced leader (SL) RNA and most protein coding mRNAs. To understand the regulation of RNA polymerase II transcription in these parasites, we have purified a transcriptionally active enzyme through affinity chromatography of its essential subunit, RPB4. The enzyme preparation is active in both promoter-independent and promoter-dependent in vitro transcription assays. Importantly, the enzyme is sensitive to alpha-amanitin inhibition, a hallmark of eukaryotic RNA polymerase II enzymes. Using mass spectrometric analysis we have identified the previously unobserved RPB12 subunit of T. brucei RNA polymerase II. TbRPB12 contains a conserved CX(2)CX(10-15)CX(2)C zinc binding motif that is characteristic of other eukaryotic RPB12 polypeptides. We also identified seven proteins that associate with T. brucei RNA polymerase II. While both bioinformatics and biochemical analysis have focused on the subunit structure of trypanosome RNA polymerases, this is the first study that reveals a functional RNA polymerase II enzyme.

Amanitins↗
PubMed1998

EWS, but not EWS-FLI-1, is associated with both TFIID and RNA polymerase II: interactions between two members of the TET family, EWS and hTAFII68, and subunits of TFIID and RNA polymerase II complexes.

The t(11;22) chromosomal translocation specifically linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the amino-terminus-encoding region of the EWS gene to the carboxyl-terminal DNA-binding domain encoded by the FLI-1 gene. As the function of the protein encoded by the EWS gene remains unknown, we investigated the putative role of EWS in RNA polymerase II (Pol II) transcription by comparing its activity with that of its structural homolog, hTAFII68. We demonstrate that a portion of EWS is able to associate with the basal transcription factor TFIID, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFIIs). In vitro binding studies revealed that both EWS and hTAFII68 interact with the same TFIID subunits, suggesting that the presence of EWS and that of hTAFII68 in the same TFIID complex may be mutually exclusive. Moreover, EWS is not exclusively associated with TFIID but, similarly to hTAFII68, is also associated with the Pol II complex. The subunits of Pol II that interact with EWS and hTAFII68 have been identified, confirming the association with the polymerase. In contrast to EWS, the tumorigenic EWS-FLI-1 fusion protein is not associated with either TFIID or Pol II in Ewing cell nuclear extracts. These observations suggest that EWS and EWS-FLI-1 may play different roles in Pol II transcription.

Animals↗
PubMed2001

c-Jun binds the N terminus of human TAF(II)250 to derepress RNA polymerase II transcription in vitro.

c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250. The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro.

Chromatography, Affinity↗
PubMed2006

Influenza virus inhibits RNA polymerase II elongation.

The influenza virus RNA-dependent RNA polymerase interacts with the serine-5 phosphorylated carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II). It was proposed that this interaction allows the viral RNA polymerase to gain access to host mRNA-derived capped RNA fragments required as primers for the initiation of viral mRNA synthesis. Here, we show, using a chromatin immunoprecipitation (ChIP) analysis, that similar amounts of Pol II associate with Pol II promoter DNAs in influenza virus-infected and mock-infected cells. However, there is a statistically significant reduction in Pol II densities in the coding region of Pol II genes in infected cells. Thus, influenza virus specifically interferes with Pol II elongation, but not Pol II initiation. We propose that influenza virus RNA polymerase, by binding to the CTD of initiating Pol II and subsequent cleavage of the capped 5' end of the nascent transcript, triggers premature Pol II termination.

Chromatin Immunoprecipitation↗
PubMed1984

Complex RNA chain elongation kinetics by wheat germ RNA polymerase II.

Kinetics of RNA chain elongation catalyzed by wheat germ RNA polymerase II have been studied using various synthetic DNA templates in the presence of excess dinucleotide monophosphate primers. With single- or double-stranded homopolymer templates, the double reciprocal plots 1/(velocity) as a function of 1/(nucleotide substrate) exhibit positive, negative or no curvature. With poly(dAT) as template, the mechanism of nucleoside monophosphate incorporation into RNA is not the ping-pong kinetic mechanism which was derived for E. coli RNA polymerase (6). Noncomplementary nucleoside triphosphates inhibit RNA transcription allosterically. Cordycepin triphosphate behaves as ATP, and not only inhibits AMP incorporation but also that of UMP and GMP on appropriate templates. The reason for this complex kinetic behavior is not yet understood. Possibilities are raised that there are several nucleoside triphosphate binding sites on wheat germ RNA polymerase II, that additional nucleoside triphosphate dependent enzymatic activities are required for reaction to occur or that the Km value for incorporation of a given nucleoside monophosphate into RNA is dependent on the length of the RNA chain and/or the nucleotide sequence surrounding the complementary base on the DNA template.

Kinetics↗
PubMed1993

Uncoupling of initiation and reinitiation rates during HeLa RNA polymerase II transcription in vitro.

RNA polymerase II transcription is influenced both by how rapidly a gene is induced and by the rate at which continuous reinitiation occurs after induction. We show here that in vitro the rates of these two critical steps need not be the same. For activator GAL-AH-dependent HeLa transcription, the rate of assembling a preinitiation complex is significantly slower than the rate of reinitiation. Although reinitiation is rapid, it still requires ATP hydrolysis. This unexpected uncoupling of the rates of initiation and reinitiation implies that in regulating mammalian promoter activity, one must consider separately the controls on initiation during induction and the controls on the subsequent reinitiation events.

Adenosine Triphosphate↗
PubMed1995

Specific binding of RNA polymerase II to the human immunodeficiency virus trans-activating region RNA is regulated by cellular cofactors and Tat.

The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression in response to Tat is dependent on an element downstream of the HIV-1 transcriptional initiation site designated the trans-activating region (TAR). TAR forms a stable stem-loop RNA structure in which a 3-nt bulge structure and a 6-nt loop structure are important for Tat activation. In the absence of Tat, the HIV-1 promoter generates so-called short or nonprocessive transcripts terminating at +60, while in the presence of Tat the synthesis of these short transcripts is markedly decreased and transcripts that extend through the 9.0-kb HIV-1 genome are synthesized. Tat effects on transcriptional elongation are likely due to alterations in the elongation properties of RNA polymerase II. In this study we demonstrated that a set of cellular cofactors that modulate the binding of the cellular protein TRP-185 to the TAR RNA loop sequences also functioned to markedly stimulate the specific binding of hypophosphorylated (IIa) and hyperphosphorylated (IIo) RNA polymerase II to TAR RNA. The concentrations of RNA polymerase II required for this interaction with TAR RNA were similar to those required to initiate in vitro transcription from the HIV-1 long terminal repeat. RNA gel retardation analysis with wild-type and mutant TAR RNAs indicated that the TAR RNA loop and bulge sequences were critical for the binding of RNA polymerase II. The addition of wild-type but not mutant Tat protein to gel retardation analysis with TAR RNA and RNA polymerase II resulted in the loss of binding of RNA polymerase II binding to TAR RNA. These results suggest that Tat may function to alter RNA polymerase II, which is paused due to its binding to HIV-1 TAR RNA with resultant stimulation of its transcriptional elongation properties.

Gene Expression Regulation, Viral↗
PubMed1987

Factors involved in specific transcription by mammalian RNA polymerase II. Transcription factor IIS stimulates elongation of RNA chains.

A factor that stimulates random transcription of purified DNAs by RNA polymerase II has been partially purified and analyzed with respect to its possible role in specific transcription from class II promoters. Studies of the effect of this factor (transcription factor IIS) on transcription from the adenovirus major late promoter in a system reconstituted with RNA polymerase II and purified factors (IIA, IIB, IIE, and IID) indicated that it acted subsequent to the initiation step and that it stimulated the rate of elongation. Kinetic experiments indicated that the factor affected the efficiency with which the RNA polymerase II passed through pausing sites. The relationship of transcription factor IIS to a protein previously purified from Erlich ascites tumor cells (Sekimizu, K., Nakanishi, Y., Mizuno, D., and Natori, S. (1979) Biochemistry 18, 1582-1588) was also studied.

Adenoviridae↗