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PubMed1998

Escherichia coli rho factor induces release of yeast RNA polymerase II but not polymerase I or III.

Purified RNA polymerase II (pol II) from the yeast Saccharomyces cerevisiae pauses without releasing at many locations during in vitro transcription. Pausing can be induced by intrinsic DNA sequence as well as by specific DNA bound proteins such as the RNA pol I termination factor, Reb1p, or lac repressor. Addition of rho termination factor from E. coli induces RNA pol II to release at all of these pause sites. Rho-induced release of pol II requires both a rho binding site in the transcript upstream of the pause sites as well as hydrolysis of ATP. In contrast, rho factor has no effect on either pausing or release by RNA pol I or III. When combined with previous observations, these results suggest that RNA pol II may terminate by a mechanism closely related to the rho-dependent mechanism of prokaryotes. In contrast, pol I and III appear to utilize a mechanism more related to the rho-independent terminators of prokaryotes.

Adenosine Triphosphate↗
PubMed1980

RNA primers and the role of host nuclear RNA polymerase II in influenza viral RNA transcription.

Influenza viral RNA transcription in the infected cell is inhibited by alpha-amanitin, a specific inhibitor of the host nuclear RNA polymerase II. Because viral RNA transcription in vitro catalysed by the virion-associated transcriptase is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by RNA polymerase II. In addition, because we did not detect any capping and methylating enzymes in virions, we have proposed that the 5' terminal methylated cap found on in-vivo viral messenger RNA (mRNA) is derived from the putative primer RNAs. Our recent experiments have proved these two hypotheses. Purified globin mRNAs were shown to stimulate viral RNA transcription in vitro very effectively. The resulting transcripts directed the synthesis of all the non-glycosylated virus-specific proteins in cell-free systems. Other eukaryotic mRNAs were also active as primers. The presence of a 5' terminal methylated cap structure in the priming mRNA was required for its priming activity. Thus, with globin mRNA, removal of the cap eliminated essentially all of its priming activity, and much of this activity could be restored by enzymically recapping the globin mRNA. Using globin mRNA containing 32P only in its cap, we demonstrated that the 5' cap of the globin mRNA primer was physically transferred to the viral RNA transcripts during transcription. Gel electrophoretic analysis suggested that, in addition to the cap, about 10-15 other nucleotides were also transferred from the globin mRNA to the viral RNA transcripts. A mechanism for the priming of influenza viral RNA transcription by globin mRNA is proposed. Initial experiments strongly suggest that priming by capped host mRNAs also occurs during the synthesis of viral mRNA in vivo.

Amanitins↗
PubMed1994

Basal components of the transcription apparatus (RNA polymerase II, TATA-binding protein) contain activation domains: is the repetitive C-terminal domain (CTD) of RNA polymerase II a "portable enhancer domain"?

Regions rich in serine, threonine, and proline residues can be found in transcriptional activation domains, as well as in the N-terminal parts of mammalian TATA-binding proteins, where they are interrupted by polyglutamine stretches. Likewise, the C-terminal domain of the largest subunit of RNA polymerase II contains multiple repeats of the consensus heptapeptide sequence YSPTSPS. To test directly for possible activation functions, we fused the GAL4 DNA-binding domain to the N-terminal domain of human TBP or subdomains of it, and to the C-terminal domain (CTD) of mouse RNA polymerase II or synthetic polymers of a CTD consensus repeat. We found that these chimeric proteins were able to activate transcription when bound to a GAL4 site in front of the TATA box, a function characteristic of transcription factors. However, while subdomains of TBP functioned only from a position close to the TATA box ("promoter" position), multiple repeats of the CTD consensus sequence were also able to mediate transcriptional activation from a remote ("enhancer") position. Our findings suggest that a region of TBP that is unique to mammals functionally cooperates with "proximal" activation domains of promoter-bound transcription factors. They also imply that the C-terminal domain of RNA polymerase II includes a function that is otherwise confined to remote activation domains of enhancer-bound transcription factors. We suggest that the CTD of RNA polymerase II contains a "portable" remote activation domain that may also facilitate chromatin opening within the transcription unit.

Amino Acid Sequence↗
PubMed1989

Drug inhibitors of RNA polymerase II transcription.

Transcription by RNA polymerase II occurs after formation of a transcription complex. This complex is assembled in stages by the interaction of transcription factors with the template and/or with each other. We report on the ability of six drugs to inhibit the assembly of the RNA polymerase II transcription complex. Assembly of the complex on the adenovirus major late promoter requires several transcription factors. The normal assembly process requires that the DNA first interact with TFIIA, then with TFIID, and finally with at least four additional transcription factors (one of which is RNA polymerase II). We observed that streptolydigin (10 micrograms/ml) inhibits association of ILA and IID, and at higher concentrations (100 micrograms/ml) inhibits that IIA/IID complex from binding to DNA. Streptovaricin (100 micrograms/ml) appears to inhibit the IIA/IID interaction with DNA and prevents reinitiation (at 500 micrograms/ml). Adriamycin (1 microgram/ml) inhibits the interaction of TFIID with the IIA/DNA complex and inhibits an additional event immediately prior to, or during, elongation. Daunorubicin may be an elongation inhibitor. Heparin at 10 micrograms/ml inhibits further assembly after the IIA/IID/DNA complex has formed, and at 100 micrograms/ml also inhibits a late event in the assembly process and blocks reinitiation. Rifamycin AF/013 (100 micrograms/ml) inhibits the early events necessary to form the IIA/IID/DNA complex and (at 10 micrograms/ml) an assembly event following formation of the IIA/IID/DNA complex. Therefore, these compounds should be useful as probes for further examination of the assembly process.

Aminoglycosides↗
PubMed1987

Studies on the mechanism of glucocorticoid hormone induced alterations in rat thymic transcription--II. Partial purification and characterization of RNA polymerases II from hydrocortisone and control vehicle treated animals.

In experiments designed to study the mechanism of glucocorticoid hormone induced reductions in rat thymic transcription, adrenalectomized rats were injected with hydrocortisone (50 mg/kg) or control vehicle 12 h prior to sacrifice. Thymic nuclei were used to prepare soluble nuclear extracts containing RNA polymerase II. Nuclear extract RNA polymerases II were then partially purified (600-fold) on DEAE-Sephadex columns and characterized. The responses of partially purified thymic RNA polymerases II from rats treated in vivo with hydrocortisone or vehicle were similar to: pH, temperature, ionic strength, trypsin proteolysis, and inhibition by alpha-amanitin; however, RNA polymerase II from hydrocortisone treated animals was consistently reduced in activity compared to control RNA polymerase II. Determination of the apparent specific activities of peak RNA polymerase II fractions from DEAE-Sephadex columns suggested that the specific activity of RNA polymerase II from hydrocortisone treated animals was reduced compared to RNA polymerase II activity from control animals. The fact that both nuclear extract and partially purified RNA polymerases II from hydrocortisone treated rats were reduced in activity when assayed in reconstituted transcriptive systems suggests a denatured, defective or modified RNA polymerase II molecule acting as a transcription inhibitor. Thermally denatured nucleoplasmic RNA polymerase II fractions were shown to interfere with transcription by native nucleoplasmic RNA polymerase II in vitro, but did not appear to inhibit transcription to he degree observed in vitro following in vivo hydrocortisone administration.

Amanitins↗
PubMed2006

Structure and dynamics of RNA polymerase II elongation complex.

RNA polymerase (Pol) II is a fundamental and important enzyme in the transcription process. However, two mysterious questions have remained unsolved: how an unwound bubble of DNA is established and maintained, and how the enzyme moves along the DNA. To answer these questions, we constructed a model structure of the Pol II elongation complex with the 50 base pairs of DNA-24 bases of RNA including the unwound bubble of DNA and performed a molecular dynamics simulation. We obtained a reliable model structure of the Pol II elongation complex in the pre-translocation state which has not yet been determined by the X-ray crystallographic study. The model structure revealed that multiple protein loops work concertedly to form and maintain the bubble structure. We also found that the conformational change of a loop in the Pol II, fork loop 1, couples with the unidirectional movement of the Pol II along the DNA.

Crystallography, X-Ray↗
PubMedDate not supplied

The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila.

The RNA polymerase II elongation complex contains several factors that facilitate transcription elongation and catalyse the processing of precursor messenger RNAs (pre-mRNAs). The conserved elongation factor Spt6 is recruited rapidly and robustly to sites of active transcription. Here we show that Drosophila Spt6 (dSpt6) co-purifies with the exosome, a complex of 3' to 5' exoribonucleases that is implicated in the processing of structural RNA and in the degradation of improperly processed pre-mRNA. Immunoprecipitation assays of Drosophila nuclear extracts show that the exosome also associates with the elongation factor dSpt5 and RNA polymerase II. In vivo, exosome subunits colocalize with dSpt6 at transcriptionally active loci on polytene chromosomes during normal development and are strongly recruited to heat-shock loci on gene induction. At higher resolution, chromatin immunoprecipitation analysis shows that the exosome is recruited to transcriptionally active units of heat-shock genes. These data provide a physical basis for the hypothesis that exosome-mediated pre-mRNA surveillance accompanies transcription elongation.

Animals↗
PubMed1994

An RNA polymerase II holoenzyme responsive to activators.

RNA polymerase II requires multiple general transcription factors to initiate site-specific transcription. These proteins can assemble in an ordered fashion onto promoter DNA in vitro, and such ordered assembly may occur in vivo (Fig. 1a). Some general transcription factors can interact with RNA polymerase II in the absence of DNA, however, suggesting that RNA polymerase II may also assemble into a multi-component complex containing a subset of initiation factors before binding to promoter DNA (Fig. 1b). Here we present evidence from the yeast Saccharomyces cerevisiae for such an RNA polymerase II holoenzyme, a multi-subunit complex containing roughly equimolar amounts of RNA polymerase II, a subset of general transcription factors, and SRB regulatory proteins. Transcription by this holoenzyme is stimulated by the activator protein GAL4-VP16, a feature not observed with purified RNA polymerase II and general transcription factors alone. We propose that the holoenzyme is a form of RNA polymerase II readily recruited to promoters in vivo.

DNA, Fungal↗
PubMed1994

Human RNA polymerase II subunit hRPB14 is homologous to yeast RNA polymerase I, II, and III subunits (AC19 and RPB11) and is similar to a portion of the bacterial RNA polymerase alpha subunit.

The cDNA cloning of the human polII 14-kDa subunit, hRPB14, and the comparison of its aa sequence with those of other pol subunits are described. The aa sequence of hRPB14 has homology to yeast poIII subunit RPB11 (44%), to a common subunit of yeast polI and polIII AC19 (24%) and to a Caenorhabditis elegans sequence (33%). hRPB14 contains a 19-aa motif, located in its N terminus, which was also found in human polII 33-kDa subunit hRPB33, yeast pol subunits (AC40, AC19, RPB3 and RPB11), and in the bacterial pol alpha subunit, which was involved in subunit assembly. This motif was also conserved in the conjugation-specific gene products of Tetrahymena (CnjC), Merchantia polymorpha chloroplast DNA (RNLVA) and C. elegans DNA (CEF58A4; deduced from the nucleotide sequence and of unknown function). The evolutionary emergence of a probable eukaryotic heterodimer, hRPB14/hRPB33, from a prokaryotic homodimer, alpha 2, is hypothesized.

Amino Acid Sequence↗
PubMed1982

Developmental genetics of a temperature-sensitive RNA polymerase II mutation in Drosophila melanogaster.

Purified RNA polymerase II (RNA nucleotidyl-transferase; EC 2.7.7.6) extracted from flies possessing lesions in the Ultrabithorax-like (Ubl) locus of Drosophila melanogaster has altered activity in vitro (Greenleaf et al. 1979, 1980; Coulter and Greenleaf 1982). This strongly suggests that the Ubl locus encodes a subunit of RNA polymerase II. Ethyl methanesulfonate was used to induce a temperature-sensitive mutation in this locus. Flies either homozygous or hemizygous for this new X-linked mutation (Ublts) display viability comparable to that of wild-type flies at 22 degrees C but are lethal at 29 degrees C. The temperature-sensitive period for Ublts flies is between gastrulation (6 h, 29 degrees C) and pupation (9-10 days, 22 degrees C). Zygotes shifted from 22 degrees C to 29 degrees C die at either the late embryonic or first larval instar stage while temperature shifts of second and third instar larvae result in the lethal phase occurring at the pupal stage. Most pupae shifted from 22 degrees C to 29 degrees C undergo metamorphosis and eclose as adults. Adults are viable if placed at 29 degrees C; however, all females and some males become sterile if maintained at this temperature. Somatic recombination was used to induce clones homozygous for a null allele of Ubl at different stages of development. Clones of this null allele appear to be cell lethal indicating that the Ubl+ gene product is required at all stages of development. The viability of Ublts pupae and adults at 29 degrees C may result from only a partial reduction in activity caused by the mutation at this nonpermissive temperature.

Alleles↗
PubMed1991

RNase-like domain in DNA-directed RNA polymerase II.

DNA-directed RNA polymerase is responsible for gene expression. Despite its importance, many details of its function and higher-order structure still remain unknown. We report here a local sequence similarity between the second largest subunit of RNA polymerase II and bacterial RNases Ba (barnase), Bi, and St. The most remarkable similarity is that the catalytic sites of the RNases are shared with the eukaryotic RNA polymerase II subunits of Drosophila melanogaster and Saccharomyces cerevisiae. Several amino acids conserved among the RNases and the RNase-like domains of the RNA polymerase subunits are located in the neighborhood of the catalytic sites of barnase, whose three-dimensional structure has been resolved. This observation suggests the functional importance of the RNase-like domain of the RNA polymerase subunits and indicates that the RNase-like domain may have RNase activity. The location of the RNase-like domain relative to the region necessary for RNA polymerization is similar to the relative proximity of 5'----3' or 3'----5' exonuclease and the region of polymerase activity of DNA polymerase I. The RNase-like domain might work in proofreading, as in RNA-directed RNA polymerase of influenza virus, or it may contribute to RNA binding through an unknown function.

Amino Acid Sequence↗
PubMed2006

Perspectives on the RNA polymerase II core promoter.

The RNA polymerase II core promoter is a critical yet often overlooked component in the transcription process. The core promoter is defined as the stretch of DNA, which encompasses the RNA start site and is typically approx. 40-50 nt in length, that directs the initiation of gene transcription. In the past, it has been generally presumed that core promoters are general in function and that transcription initiation occurs via a common shared mechanism. Recent studies have revealed, however, that there is considerable diversity in core promoter structure and function. There are a number of DNA elements that contribute to core promoter activity, and the specific properties of a given core promoter are dictated by the presence or absence of these core promoter motifs. The known core promoter elements include the TATA box, Inr (initiator), BRE(u) {BRE [TFIIB (transcription factor for RNA polymerase IIB) recognition element] upstream of the TATA box} and BRE(d) (BRE downstream of the TATA box), MTE (motif ten element), DCE (downstream core element) and DPE (downstream core promoter element). In this paper, we will provide some perspectives on current and future issues that pertain to the RNA polymerase II core promoter.

Models, Genetic↗
PubMed1985

A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II.

Purified eukaryotic nuclear RNA polymerase II consists of three subspecies that differ in the apparent molecular masses of their largest subunit, designated IIo, IIa, and IIb for polymerase species IIO, IIA, and IIB, respectively. Subunits IIo, IIa, and IIb are the products of a single gene. We present here the amino acid composition of calf thymus subunits IIa and IIb and the C-terminal amino acid sequence of subunit IIa (IIo) inferred from the nucleotide sequence of part of the mouse gene encoding this RNA polymerase subunit. The calculated amino acid composition of the peptide unique to subunit IIa indicates that subunit IIa contains a domain rich in serine, proline, threonine, and tyrosine. The sequence at the 3' end of the mouse RNA polymerase II largest subunit gene reveals that the C-terminal domain consists of 52 repeats of a seven amino acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This sequence is also unusual in that it contains a high percentage of potential phosphorylation sites.

Amino Acid Sequence↗
PubMed2001

The 3' Igkappa enhancer contains RNA polymerase II promoters: implications for endogenous and transgenic kappa gene expression.

We have created a kappa transgene in which a polymerase (pol) III promoter replaces the pol II promoter. Two independent transgenic lines show somatic hypermutation of the transgene in B cells from hyperimmunized mice. Both lines transcribe transgenes from the pol III promoter in the liver. However, in spleen and spleen B cell-derived hybridomas, they also transcribe mRNA from pol II promoters located within the 3' kappa enhancer of the preceding transgene copy in a tandem transgene array. The findings demonstrate that in an array of multiple transgenes the expression (and somatic hypermutation) of an individual transgene copy must be considered in the context of the other copies. We also show that sequences around the 3' kappa enhancer in endogenous genes are transcribed. The possible role of these promoters in endogenous kappa gene expression is discussed. An unrelated finding in this study was a novel RNA splice in one hybridoma.

Animals↗
PubMed1997

Glycosylation of RNA polymerase II from wheat germ.

RNA polymerase II from wheat germ was analyzed for the presence of sugars. The two largest subunits and the 27 and 25 kDa subunits were found to be glycosylated by a variety of sugars. However, no N-acetylglucosamine was detected, which was found by Kelly et al. (J. Biol. Chem. (1993) 268, 10416-10424) in the largest subunit of RNA polymerase II from calf thymus. Thus it appears that the regulatory function of this sugar, postulated by Kelly et al., is performed in the wheat germ enzyme by other monosaccharides. Carbohydrate analysis of the two largest subunits of the calf thymus enzyme also revealed the presence, beside N-acetylglucosamine, of other sugars. Some similarities in the features of glycosylation of the two polymerases, isolated from very different organisms, suggest that the sugar moieties have an important role in the structure and/or function of these enzymes.

Animals↗
PubMed1999

A role for the TFIIH XPB DNA helicase in promoter escape by RNA polymerase II.

TFIIH is an RNA polymerase II transcription factor that performs ATP-dependent functions in both transcription initiation, where it catalyzes formation of the open complex, and in promoter escape, where it suppresses arrest of the early elongation complex at promoter-proximal sites. TFIIH possesses three known ATP-dependent activities: a 3' --> 5' DNA helicase catalyzed by its XPB subunit, a 5' --> 3' DNA helicase catalyzed by its XPD subunit, and a carboxyl-terminal domain (CTD) kinase activity catalyzed by its CDK7 subunit. In this report, we exploit TFIIH mutants to investigate the contributions of TFIIH DNA helicase and CTD kinase activities to efficient promoter escape by RNA polymerase II in a minimal transcription system reconstituted with purified polymerase and general initiation factors. Our findings argue that the TFIIH XPB DNA helicase is primarily responsible for preventing premature arrest of early elongation intermediates during exit of polymerase from the promoter.

Adenosine Triphosphate↗
PubMed1990

RNA polymerase II: subunit structure and function.

RNA polymerase II is the core of the complex apparatus that is responsible for the regulated synthesis of mRNA. A comprehensive knowledge of RNA polymerase II is essential to our understanding of the molecular mechanisms through which a variety of transcription factors regulate eukaryotic gene expression. The recent cloning of genes for all ten subunits of yeast RNA polymerase II has revealed intriguing similarities and differences between the eukaryotic RNA polymerase and its simpler prokaryotic counterpart. Epitope tagging and other experiments made possible by the cloning of these genes have provided a clearer picture of RNA polymerase II subunit composition, stoichiometry and function, and set the stage for further investigating the dialogue between RNA polymerase II and transcription factors.

Amino Acid Sequence↗