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Results for “RNA Polymerase II”

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PubMed1982

Non-processive transcription of poly[d(A-T)] by wheat germ RNA polymerase II.

RNA product distribution obtained during the transcription of poly[d(A-T)] by wheat germ RNA polymerase IIA under various experimental conditions was analyzed by high resolution polyacrylamide gel electrophoresis. Poly[r(A-U)] synthesis proceeded as if wheat germ RNA polymerase II was a non-processive enzyme: a ladder of RNA products of increasing lengths was obtained, which apparently, terminated at every other nucleotide. RNA release was not dependent upon nucleoside triphosphate substrate concentrations. A likely explanation would be that ternary complexes enzyme: DNA: RNA were very much unstable; moreover, oligonucleotides released were not re-used for further elongation by the enzyme.

DNA-Directed RNA Polymerases↗

PubMed2001

Roles of RNA:DNA hybrid stability, RNA structure, and active site conformation in pausing by human RNA polymerase II.

Human RNA polymerase II recognizes a strong transcriptional pause signal in the initially transcribed region of HIV-1. We report the use of a limited-step transcription assay to dissect the mechanism underlying recognition of and escape from this HIV-1 pause. Our results suggest that the primary determinant of transcriptional pausing is a relatively weak RNA:DNA hybrid that triggers backtracking of RNA polymerase II along the RNA and DNA chains and displaces the RNA 3' OH from the active site. In contrast, two alternative RNA secondary structures, TAR and anti-TAR, are not required for pausing and affect it only indirectly, rather than through direct interaction with RNA polymerase II. TAR accelerates escape from the pause, but anti-TAR inhibits formation of TAR prior to pause escape. The behavior of RNA polymerase II at a mutant pause signal supports a two-step, non-equilibrium mechanism in which the rate-determining step is a conformational change in the enzyme, rather than the changes in nucleic-acid base-pairing that accompany backtracking.

Base Pairing↗

PubMed1976

DNA-dependent RNA polymerases from Artemia salina. Subunit structure of polymerase II.

RNA polymerase II from larvae of the brine shrimp, Artemia salina, was highly purified by two cycles of DEAE-cellulose chromatography followed by centrifugation through discontinuous sucrose gradients. Gradient fractions were subjected to elctrophoresis is polyacrylamide gels containing sodium dodecyl sulfate. The subunit structure of RNA polymerase II was determined by quantitative comparison of the polypeptides and enzyme activity present in each gradient fraction. The enzyme contains one copy of each of four subunits with estimated molecular weights of 170,000, 130,000, 36,000 and 24,000. The total molecular weight agrees well with the molecular weight estimated for the native enzyme by density gradient centrifugation.

DNA-Directed RNA Polymerases↗

PubMed1995

Association of an activator with an RNA polymerase II holoenzyme.

RNA polymerase II holoenzymes have been described that consist of RNA polymerase II, a subset of general transcription factors, and four SRB proteins. The SRB proteins, which were identified through a selection for genes involved in transcription initiation by RNA polymerase II in vivo, are a hallmark of the holoenzyme. We report here the isolation and characterization of additional SRB genes. We show that the products of all nine SRB genes identified thus far are components of the RNA polymerase II holoenzyme and are associated with a holoenzyme subcomplex termed the mediator of activation. The holoenzyme is capable of responding to a transcriptional activator, suggesting a model in which activators function, in part, through direct interactions with the holoenzyme. Immunoprecipitation experiments with anti-SRB5 antibodies demonstrate that the acidic activating domain of VP16 specifically binds to the holoenzyme. Furthermore, the holoenzyme and the mediator subcomplex bind to a VP16 affinity column. These results provide a more complete description of the RNA polymerase II holoenzyme and suggest that this form of the transcription apparatus can be recruited to promoters via direct interactions with activators.

Amino Acid Sequence↗

PubMed2006

Nuclear export of influenza A virus mRNAs requires ongoing RNA polymerase II activity.

Influenza A virus transcribes its segmented negative sense RNA genome in the nuclei of infected cells in a process long known to require host RNA polymerase II (RNAP-II). RNA polymerase II synthesizes pre-mRNAs whose 5'-cap structures are scavenged by the viral RNA-dependent RNA polymerase during synthesis of viral mRNAs. Drugs that inhibit RNAP-II therefore block viral replication, but not necessarily solely by denying the viral polymerase a source of cap-donor molecules. We show here that 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), a compound that prevents processive transcription by RNAP-II, inhibits expression of the viral HA, M1 and NS1 genes at the post-transcriptional level. Abundant quantities of functionally and structurally intact viral mRNAs are made in the presence of DRB but with the exception of NP and NS2 mRNAs, are not efficiently translated. Taking M1 and NP mRNAs as representatives of DRB-sensitive and insensitive mRNAs, respectively, we found that the block to translation operates at the level of nuclear export. Furthermore, removal of DRB reversed this block unless a variety of chemically and mechanistically distinct RNAP-II inhibitors were added instead. We conclude that influenza A virus replication requires RNAP-II activity not just to provide capped mRNA substrates but also to facilitate nuclear export of selected viral mRNAs.

Active Transport, Cell Nucleus↗

PubMed2002

N4 RNA polymerase II, a heterodimeric RNA polymerase with homology to the single-subunit family of RNA polymerases.

Bacteriophage N4 middle genes are transcribed by a phage-coded, heterodimeric, rifampin-resistant RNA polymerase, N4 RNA polymerase II (N4 RNAPII). Sequencing and transcriptional analysis revealed that the genes encoding the two subunits comprising N4 RNAPII are translated from a common transcript initiating at the N4 early promoter Pe3. These genes code for proteins of 269 and 404 amino acid residues with sequence similarity to the single-subunit, phage-like RNA polymerases. The genes encoding the N4 RNAPII subunits, as well as a synthetic construct encoding a fusion polypeptide, have been cloned and expressed. Both the individually expressed subunits and the fusion polypeptide reconstitute functional enzymes in vivo and in vitro.

Amino Acid Sequence↗

PubMed1990

Analysis of the signals for transcription termination by purified RNA polymerase II.

Eukaryotic RNA polymerase II recognizes certain DNA sequences as effective signals for transcription termination in vitro. Previously, we have shown that such termination occurs within T-rich sequences; however, not all T runs stop the enzyme nor is the efficiency of termination correlated with the length of the T run. Here we have investigated the sequence elements that signal transcription termination by purified RNA polymerase II. We have examined terminators located within introns of the human histone H3.3 gene and the human c-myc gene. Deletion analysis of the H3.3 termination region indicates that the sequences between -6 and +24 relative to the strongest termination site are sufficient to cause transcription termination. The minimal termination signal at this site has been localized to the sequence TTTTTTTC-CCTTTTTT in the nontranscribed strand. A similar but nonidentical sequence has been defined for the c-myc termination site. Since RNA polymerase II terminates transcription only within the first run of T residues in these sequences, at least part of the termination signal lies in downstream nontranscribed DNA sequences. Restriction fragment mobility analysis indicates that the H3.3 termination region contains a bend in the DNA helix. Oligonucleotides containing the minimal termination signals also cause restriction fragments to migrate with anomalous mobility. A region of the SV40 genome containing a previously characterized bend also causes RNA polymerase II to terminate transcription. We suggest that a structural element causing a bend in the DNA helix may be part of the signal for transcription termination by purified RNA polymerase II.

Animals↗

PubMed1989

Promoter specificity and modulation of RNA polymerase II transcription.

RNA polymerase II is a multisubunit enzyme involved in the transcription of protein encoding genes. Recently acquired knowledge of the transcription process and of the RNA polymerase molecule as well as the isolation of subunit clones have led to a better understanding of the enzyme's functional regulation. Specific transcription initiation occurs at promoter regions located upstream of the gene and requires a minimum of five basic factors in addition to the enzyme. Furthermore, proteins that bind to specific DNA elements within the promoter also regulate transcriptional activity. Additional factors are required for the elongation and, possibly, termination of transcription. Two elongation factors, SII and TFIIF, interact directly with the RNA polymerase II molecule. Functional domains of RNA polymerase II have been determined by analysis of genomic clones for the two largest subunits of the enzyme. For example, the 240-kDa largest subunit contains a highly phosphorylated carboxyl-terminal heptapeptide domain repeated 26-52 times that is absolutely required for transcription in vivo. Analysis of the polymerase molecule and its interaction with basic gene-specific transcription factors will aid in our studies of the control of gene expression.

Amino Acid Sequence↗

PubMed1984

Dinucleotide priming of transcription mediated by RNA polymerase II.

Mammalian RNA polymerase II was shown to utilize dinucleoside monophosphates for priming of promoter specific RNAs. In a reconstituted system containing purified polymerase and HeLa cell fractions, dinucleotides were incorporated by complementarity with template sequences at the in vivo cap sites of the adenovirus major late and adenovirus early region IV promoters. Incorporation was shown by label transfer experiments and by determining the size of 5'-terminal RNase T1-resistant oligonucleotides. All 16 dinucleotides were tested for priming of RNA chains at the major late promoter. RNA polymerase II initiated with various primers over a contiguous region of 9 bases, centered around the in vivo initiation site. We suggest that the polymerase drifts or oscillates over this region. Using a dinucleotide challenge protocol, the rate of initiation at the major late promoter was measured following preincubation of the template DNA with RNA polymerase II and factors. Initiation with ATP was 90% complete within the 1st min after addition of nucleotide triphosphates. Stimulation of transcription by dinucleotides was not observed, due to this rapid initiation. The 5'-hydroxyl terminus of dinucleotide-primed RNAs remained unmodified. Although transcripts initiated with ATP were rapidly capped in whole cell extracts, ATP-primed RNA synthesized in the reconstituted system retained free 5'-terminal phosphates. Thus, capping was not essential for synthesis of long runoff RNAs.

Adenosine Triphosphate↗

PubMed1987

Functional redundancy and structural polymorphism in the large subunit of RNA polymerase II.

The RNA polymerase II large subunit contains tandem copies of the sequence Pro Thr Ser Pro Ser Tyr Ser at its carboxyl terminus, the number of which varies from 26 in yeast to 52 in mice. Our results indicate that the heptapeptide repeat sequence is unique and essential to RNA polymerase II. We have determined that a portion of the heptapeptide repeat domain is essential for viability by constructing and analyzing unidirectional deletions of the carboxy-terminal coding sequence in yeast. Cells containing an RNA polymerase II large subunit with less than 10 complete heptapeptide repeats are inviable, those containing 10-12 complete repeats are conditionally viable, and those with 13 or more complete repeats are unconditionally viable. The inviable deletion mutants studied here have truncated RNA polymerase subunits that are stable, but functionally deficient. Finally, the number of repeat units is polymorphic in wild-type yeast strains. These results have implications for the function of this unusual sequence in transcription.

Amino Acid Sequence↗

PubMed1975

Separation of lymphocyte chromatin into template-active fractions with specificity for eukaryotic RNA polymerase II or prokaryotic RNA polymerase.

When chromatin prepared from WI-L2 lymphocytes by low salt extraction and shearing is centrifuged on a glycerol gradient, one area of the gradient yields chromatin enriched in template activity for Escherichia coli DNA-dependent RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase) as compared to Saccharomyces cerevisiae RNA polymerase II (or B). Another area yields chromatin preferred by the eukaryotic enzyme. Kinetic studies indicate that the differences in activity cannot be explained by differences in affinity of the enzymes for the various templates. The DNA isolated from either fraction has a molecular weight of 8.5 X 106. The "yeast active" fraction seems enriched in proteins. Mixing experiments indicate that the yeast enzyme does not alter the template in such a way as to improve it for the bacterial enzyme.

Cell Line↗

PubMed1990

Tails of RNA polymerase II.

Eukaryotic RNA polymerase II contains two distinct structural domains: a catalytic core consisting of subunits that are homologous to other multisubunit RNA polymerases, and a unique extension of the carboxy-terminus of the largest subunit comprising tandem repeats of the seven amino acid sequence YSPTSPS. This repetitive 'tail' domain is essential for polymerase function in vivo. Although the nature of this essential function is unknown, actively transcribing RNA polymerase II is known to be multiphosphorylated on this repetitive domain.

Amino Acid Sequence↗

PubMed1978

Effect of hydrostatic pressure on isolated cardiac nuclei: Stimulation of RNA polymerase II activity.

RNA polymerase activity was measured in isolated cardiac nuclei subjected to hydrostatic pressure. After 20 min of pressure, Mn2+ stimulated RNA polymerase II activity was increased. The response to pressure was inhibited by low concentrations of alpha-amanitin (1.1 microgram.cm-3) an inhibitor of polymerase II activity. The data show that pressure applied to isolated nuclei stimulates RNA polymerase II activity, forming mRNA, and suggests that direct application of pressure to cardiac nuclei may be the stimulus which triggers the augmented protein synthesis seen in pressure overload.

Amanitins↗

PubMed2001

Transcription organization and mRNA levels of the genes for all 12 subunits of the fission yeast RNA polymerase II.

The RNA polymerase II (Pol II) of eukaryotes is composed of 12 subunits, of which five are shared among Pol I, Pol II and Pol III. At present, however, little is known about the regulation of synthesis and assembly of the 12 Pol II subunits. To obtain an insight into the regulation of synthesis of these 12 Pol II subunits, Rpb1 to Rpb12, in the fission yeast Schizosaccharomyces pombe, we analysed the transcriptional organization of the rpb genes by use of the oligo capping method, and determined mRNA levels by quantitative competitive PCR assay. The intracellular concentrations of the 12 Rpb subunits in growing S. pombe cells are different, within a range of 15-fold difference between the least abundant Rpb3 and the most abundant Rpb12. The transcription of one group of genes including rpb3, rpb4, rpb5, rpb6, rpb7 and rpb10 is mainly initiated at a single site, while that of the other group of genes for rpb1, rpb2, rpb8, rpb9, rpb11 and rpb12 is initiated at multiple sites. The promoters of the first group of genes contain the TATA box sequence between -26 and -62, while the second group of genes carry TATA-less promoters. Several common sequence segments, tentatively designated 'Rpb motifs', were identified in the promoter regions of the rpb genes. Competitive PCR analysis indicated that mRNAs for Rpb1, Rpb3, Rpb7 and Rpb9 were among the group which had a low abundance, while the levels of Rpb6 and Rpb10 mRNAs were about fivefold, and that of Rpb2 mRNA was about 40-fold higher than the Rpb3 mRNA level. The levels of rpb mRNAs do not correlate with those of Rpb proteins. The protein-to-mRNA ratio or the translation efficiency is low for the rpb1, rpb2, rpb3 and rpb11 genes, encoding the homologues of subunits beta', beta, alpha and alpha, respectively, of the prokaryotic RNA polymerase core enzyme.

Base Sequence↗

PubMed1987

Protein kinase C phosphorylates leukemia RNA polymerase II.

Purified RNA polymerase II from chicken leukemia cells was found to be an effective substrate for protein kinase C but not cAMP-dependent protein kinase. Protein kinase C catalyzed the incorporation of 1-2 mol of phosphate per mol of polymerase II and the reaction was totally calcium and lipid dependent. Electrophoresis studies revealed a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 220 KDa, 180 KDa and 150 KDa, with a preferential phosphorylation of the 180 KDa polypeptide. The phosphorylated enzyme has a preference for using single-stranded DNA as the template for transcription, including transcription of the single-stranded myb oncogene sequence. Phosphoamino acid analysis indicated that both serine and threonine residues were phosphorylated at equal amounts. Phosphorylation by protein kinase C increased the affinity of substrate-polymerase binding and the initial rate of RNA synthesis, suggesting a mechanism by which gene expression can be activated by protein kinase C.

Amino Acids↗

PubMed1994

Complexes of yeast RNA polymerase II and RNA are substrates for TFIIS-induced RNA cleavage.

In the absence of DNA, purified yeast RNA polymerase II can bind RNA to form a binary complex. RNA in such RNA-RNA polymerase complexes undergoes reactions previously thought to be unique to nascent RNA in ternary complexes with DNA, including TFIIS-dependent cleavage and elongation by 3'-terminal addition of NMP from NTP. Both of these reactions are inhibited by alpha-amanitin. Hence, by several criteria the RNA in binary complexes is bound to the polymerase in a manner quite similar to that in ternary complexes in which the catalytic site for nucleotide addition is positioned at or near the 3'-OH terminus of the RNA. These findings are consistent with a model for the RNA polymerase ternary complex in which the RNA is bound at the 3' terminus through two protein-binding sites located up to 10 nt apart.

Amanitins↗

PubMed2004

Targeting of Swi/Snf to the yeast GAL1 UAS G requires the Mediator, TAF IIs, and RNA polymerase II.

The chromatin remodeling activity of the Swi/Snf complex is essential for the expression of several yeast genes. Previous studies have suggested that recruitment of Swi/Snf requires the action of transcriptional activators. However, reports in metazoans and in yeast have provided evidence of interactions between Swi/Snf and the RNA polymerase II holoenzyme/Mediator complex. Here we show that recruitment of Swi/Snf to the galactose-inducible gene GAL1 cannot be fully achieved without the integrity of the Mediator complex, TAF IIs, and RNA polymerase II. Moreover, artificial recruitment of Mediator is sufficient to tether both Swi/Snf and SAGA to the GAL1 UAS G. We further demonstrate that Swi/Snf recruitment at GAL1 does not require acetylation of chromatin by Gcn5 nor the presence of SAGA. Based on these results, we conclude that interactions between the Gal4 activator and Swi/Snf are not sufficient to recruit the latter to the GAL1 UAS G, since interactions with the Mediator, TAF IIs, and RNA polymerase II are also important.

Chromatin↗

PubMed2005

Human hHpr1/p84/Thoc1 regulates transcriptional elongation and physically links RNA polymerase II and RNA processing factors.

Cotranscriptional loading of RNA processing factors onto nascent RNA facilitates efficient gene expression. Mechanisms responsible for coupling transcription and RNA processing are not well defined, but the Saccharomyces cerevisiae TREX complex provides an example. TREX is composed of the subcomplex THO that associates with RNA polymerase II and is required for normal transcriptional elongation. THO associates with proteins involved in RNA splicing and export to form the larger TREX complex. Hence, assembly of TREX physically couples transcriptional elongation with RNA processing factors. Whether metazoan species with long, intron-containing genes utilize a similar mechanism has not been established. Here we show that human hHpr1/p84/Thoc1 associates with elongating RNA polymerase II and the RNA splicing and export factor UAP56 in intact cells. Depletion of hHpr1/p84/Thoc1 causes transcriptional elongation defects and associated cellular phenotypes similar to those observed in THO-deficient yeast. We conclude that hHpr1/p84/Thoc1 regulates transcriptional elongation and may participate in a protein complex functionally analogous to yeast TREX, physically linking elongating RNA polymerase II with RNA processing factors.

Cell Cycle Proteins↗