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PubMed1977

Transcription of yeast DNA by homologous RNA polymerases I and II: selective transcription of ribosomal genes by RNA polymerase I.

Purified yeast DNA was transcribed by homologous RNA polymerases I and II and Escherichia coli RNA polymerase. Transcripts synthesized in vitro were analyzed by molecular hybridization with complementary DNA (cDNA) synthesized from yeast poly(A)-containing mRNA with viral reverse transcriptase and ribosomal DNA labeled in vitro by nick translation with E. coli DNA polymerase I. RNA synthesized by polymerase I and II in the presence of Mn2+ contained sequences complementary to cDNA and rDNA at a frequency consistent with random transcription of the template. Similarly, E. coli RNA polymerase synthesized an apparently random transcript in the presence of either Mn2+ or Mg2+. In contrast to these results, RNA polymerase I but not polymerase II transcripts were markedly enriched in sequences complementary to rDNA when transcription was carried out in the presence of Mg2+. The observed enrichment was 15-30-fold higher than observed for polymerase II or E. coli polymerase transcripts and is consistent with the transcript being comprised of 6-10% ribosomal sequences. These data strongly suggest that RNA polymerase I plays a critical role in selective transcription of ribosomal cistrons.

DNA-Directed RNA Polymerases↗
PubMed1977

DNA-dependent RNA polymerases from Artemia salina. IV. appearance of nuclear RNA polymerase activity during pre-emergence development of encysted embryos.

Dessicated and encysted gastrulae of the brine shrimp Artemia salina remain metabolically dormant until they are rehydrated. At this time development resumes, culminating in the hatching of free swimming nauplius larvae. The resumption of embryogenesis provides a convenient system for studying biochemical events which accompany development of a eukaryotic organism, and in particular Artemia has proven useful for studies of the transcriptional regulation of gene expression. Encysted gastrulae of Artemia yielded only trace amounts of DNA-dependent RNA polymerase activity when crude nuclear pellets were subjected to sonication at high ionic strength. Furthermore, when crude nuclear pellets from encysted gastrulae and developing nauplius larvae were mixed prior to sonication, subsequent solubilization of proteins from the mixture did not yield RNA polymerase activity; sonication of the pellet from nauplii alone resulted in the solubilization of large quantities of RNA polymerases I and II as we have previously found [1]. RNA polymerases I and II were detectable in sonicates of crude nuclear pellets after 1-h incubation of Artemia cysts in sea water. This presents the possibility that dormant gastrulae of the brine shrimp contain RNA polymerase which is inactive, and that the rapid appearance of nuclear enzymatic activity which accompanies the resumption of development may not require de novo synthesis of the polymerase.

Animals↗
PubMed1985

Sequence and analysis of the gene for bacteriophage T3 RNA polymerase.

The RNA polymerases encoded by bacteriophages T3 and T7 have similar structures, but exhibit nearly exclusive template specificities. We have determined the nucleotide sequence of the region of T3 DNA that encodes the T3 RNA polymerase (the gene 1.0 region), and have compared this sequence with the corresponding region of T7 DNA. The predicted amino acid sequence of the T3 RNA polymerase exhibits very few changes when compared to the T7 enzyme (82% of the residues are identical). Significant differences appear to cluster in three distinct regions in the amino-terminal half of the protein. Analysis of the data from both enzymes suggests features that may be important for polymerase function. In particular, a region that differs between the T3 and T7 enzymes exhibits significant homology to the bi-helical domain that is common to many sequence-specific DNA binding proteins. The region that flanks the structural gene contains a number of regulatory elements including: a promoter for the E. coli RNA polymerase, a potential processing site for RNase III and a promoter for the T3 polymerase. The promoter for the T3 RNA polymerase is located only 12 base pairs distal to the stop codon for the structural gene.

Amino Acid Sequence↗
PubMed1993

Yeast RNA polymerase II subunit RPB11 is related to a subunit shared by RNA polymerase I and III.

The characterization of RNA polymerase subunit genes has revealed that some subunits are shared by the three nuclear enzymes, some are homologous, and some are unique to RNA polymerases I, II, or III. We report here the isolation and characterization of the yeast RNA polymerase II subunit RPB11, which is encoded by a single copy RPB11 gene located directly upstream of the topoisomerase I gene, TOPI, on chromosome XV. The sequence of the gene predicts an RPB11 subunit of 120 amino acids (13,600 daltons), only two amino acids shorter than the RPB9 polypeptide, that co-migrates with RPB11 under most SDS-PAGE conditions, RPB11 was found to be an essential gene that encodes a protein closely related to an essential subunit shared by RNA polymerases I and III, AC19. RPB11 contains a 19 amino acid segment found in three other yeast RNA polymerase subunits and the bacterial RNA polymerase subunit alpha. Some mutations that affect RNA polymerase assembly map within this segment, suggesting that this region may play a role in subunit interactions. As the isolation of RPB11 completes the isolation of known yeast RNA polymerase II subunit genes, we briefly summarize the salient features of these twelve genes and the polypeptides that they encode.

Amino Acid Sequence↗
PubMed1979

Expression of RNA polymerase and ribosome component genes in Escherichia coli mutants having conditionally defective RNA polymerases.

The expression of the genes coding for the beta and beta' subunits of RNA polymerase, ribosomal RNA, ribosomal proteins, and beta-galactosidase was investigated in strains carrying conditionally lethal mutations affecting either RNA polymerase core assembly or RNA polymerase enzyme activity. The mutant strain XH56 produces a temperature-sensitive beta' subunit and at 42 degrees C is defective in RNA chain initiation; consequently, little or no transcription occurs at the restrictive temperature. A partial restriction, produced by shifting the strain to 39 degrees C, resulted in a rapid fivefold increase in the transcription of the rpoB and C genes and in the synthesis of the beta- and beta'-subunit proteins for which they code. The RNA polymerase assembly-defective strains A2R7 and TS4 exhibited a 1.5- to 2-fold increase in the transcription of the rpoB and C genes and in the synthesis of beta- and beta-subunit proteins after prolonged restriction. These results demonstrate (i) that regulation of the synthesis of the beta- and beta-RNA polymerase subunits is under these conditions primarily transcriptional rather than translational, and (ii) that a stimulation of rpoB and C gene expression results from a restriction on RNA synthesis caused by either RNA polymerase inactivation or inhibition of its assembly. During restriction of the mutant strains, the transcription of the ribosome component genes exhibited patterns which were similar to transcription of the rpoB and C genes, supporting the evidence that genes coding for RNA polymerase are cotranscribed with ribosomal protein genes; transcription of the lacZ gene was observed to decrease concomitant with the stimulation of the rpoB and C genes.

Bacterial Proteins↗
PubMed1994

Cleavage of the nascent transcript induced by TFIIS is insufficient to promote read-through of intrinsic blocks to elongation by RNA polymerase II.

RNA polymerases encounter a variety of types of blocks to elongation during transcription in eukaryotic cells. At least one protein, TFIIS, can promote read-through of many types of blocks to elongation by RNA polymerase II, and this protein stimulates cleavage of the nascent transcript in stalled elongation complexes as a prelude to read-through. The C-terminal half of the TFIIS protein is sufficient for stimulating the cleavage and read-through reactions in vitro. To study how TFIIS changes the response of RNA polymerase II elongation complexes to such blocks, targeted amino acids in the C terminus of HeLa TFIIS were mutated to alanines. Two mutant TFIIS proteins as well as the unmutated C-terminal half of the TFIIS protein were purified following overexpression in Escherichia coli. Each protein was examined for read-through activity and ability to stimulate transcript cleavage in ternary elongation complexes. Mutant TFIIS5 (E174A, E175A) was reduced in read-through and cleavage activities relative to the unmutated, truncated TFIIS (delta TFIIS). Mutant TFIIS7 (K187A, K189A) was able to stimulate cleavage nearly at the rate and to the extent of the TFIIS5 mutant. In contrast to what was observed with TFIIS5, no detectable read-through was observed in the presence of the TFIIS7 mutant during the course of the reaction. Thus, there is no simple, direct correlation between the ability of TFIIS to promote cleavage and its ability to promote read-through by RNA polymerase II. These results suggest that although TFIIS is necessary to mediate the cleavage reaction that precedes the read-through event, the cleavage event itself is not sufficient to allow read-through by RNA polymerase II.

Amino Acid Sequence↗
PubMed1996

Influenza A virus RNA-dependent RNA polymerase: analysis of RNA synthesis in vitro.

Influenza A virus RNA-dependent RNA polymerase, purified from virion ribonucleoprotein particles and from which endogenous genomic RNA (vRNA) has been depleted by treatment with micrococcal nuclease, was used to study transcription initiation, elongation, and termination in vitro. Templates that contained either minus- or plus-sense influenza virus nucleoprotein minigenes with conserved 5' and 3' termini and the uridylate tract were constructed. The dinucleotide ApG and alfalfa mosaic virus RNA4 (AlMV4) were used as primers. ApG primed the synthesis of full-length positive-strand or cRNA products and shorter transcripts, depending upon the molar ratio between the nucleoprotein and the vRNA template. Sequence analysis of the ends of these transcripts demonstrated that the 5' termini of both transcripts and the 3' terminus of the full-length product were complementary to the 3' and 5' termini of the vRNA template, respectively, whereas the 3' terminus of the incomplete product corresponded to a sequence located 40 bases downstream from the 5' terminus of the template and was about 20 nucleotides downstream from the uridylate tract, which is the putative signal for polyadenylation. Binding of the cap structure of AlMV4 by the polymerase activated RNA synthesis by ligation-elongation of small genomic RNA fragments which were likely derived from a genome segment protected by the polymerase from micrococcal nuclease digestion. The sequence of these fragments mapped to a region 14 to 28 nucleotides upstream of the 3' terminus of the viral genome. Polymerase subunit involvement in transcription initiation with ApG or AlMV4 was characterized by studying the effect of purified polyclonal antisubunit immunoglobulins of the G class (IgGs) in transcription assays. These results showed that anti-PB2 IgG inhibited transcription initiation in both ApG- and AlMV4-primed reactions, whereas anti-PB1 antibodies also blocked transcription initiated with AlMV4. The differences observed in product size, product sequence, and differential inhibition by antisubunit IgGs are discussed. These observations would support the notion that the influenza virus RNA-dependent RNA polymerase undergoes a conformational change after the binding of the cap structure of host cell heterogeneous nuclear RNA by PB2, which then usually leads to endonucleolytic cleavage of the capped primer 13 nucleotides downstream from the cap.

Animals↗
PubMed2002

Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity.

Hepatitis C virus (HCV) NS5B is RNA-dependent RNA polymerase (RdRP), the essential catalytic enzyme for HCV replication. Recently, NS5A has been reported to be important for the establishment of HCV replication in vitro by the adaptive mutations, although its role in viral replication remains uncertain. Here we report that purified bacterial recombinant NS5A and NS5B directly interact with each other in vitro, detected by glutathione S-transferase (GST) pull-down assay. Furthermore, complex formation of these proteins transiently coexpressed in mammalian cells was detected by coprecipitation. Using terminally and internally truncated NS5A, two discontinuous regions of NS5A (amino acids 105-162 and 277-334) outside of the adaptive mutations were identified to be independently essential for the binding both in vivo and in vitro (Yamashita, T., Kaneko, S., Shirota, Y., Qin, W., Nomura, T., Kobayashi, K., and Mkyrakami, S. (1998) J. Biol. Chem. 273, 15479-15486). We previously examined the effect of His-NS5A on RdRP activity of the soluble recombinant NS5Bt in vitro (see Yamashita et al. above). Wild NS5A weakly stimulated at first (when less than 0.1 molar ratio to NS5B) and then inhibited the NS5Bt RdRP activity in a dose-dependent manner. The internal deletion mutants defective in NS5B binding exhibited no inhibitory effect, indicating that the NS5B binding is necessary for the inhibition. Taken together, our results support the idea that NS5A modulates HCV replication as a component of replication complex.

Animals↗
PubMed1983

Multiple forms of DNA-dependent RNA polymerases in Xenopus laevis. Properties, purification, and subunit structure of class III RNA polymerases.

The class III RNA polymerases present in Xenopus laevis tissues were solubilized and partially purified by ion exchange chromatography. Single chromatographic species were detected in extracts from ovary, liver, and a kidney-derived cultured cell, whereas two chromatographic forms were detected in liver extracts. All class III enzymes analyzed exhibited characteristic responses to ionic strength changes, synthetic polynucleotide templates, and alpha-amanitin. These chromatographic and catalytic properties readily distinguished all the class III enzymes from the corresponding class I and II enzymes but clearly failed to reveal any significant differences between the various ovarian and somatic class III enzymes (with the possible exception of one of the liver enzyme forms). The ovarian RNA polymerase III was completely soluble in low ionic strength buffers and was subjected to further purification via standard procedures involving differential centrifugation, ammonium sulfate fractionation, and ion exchange chromatography. Polyacrylamide gel electrophoresis under denaturing conditions revealed at least 10 distinct polypeptides (designated subunits a-j) which were consistently present in near equimolar amounts and which ranged in size from 155,000 to 19,000 daltons. Several smaller polypeptides (less than 19,000) were also evident in all preparations but were not further characterized. Analysis of the chromatographically purified RNA polymerase on nondenaturing gels revealed two electrophoretic forms, although subsequent analysis on denaturing gels failed to reveal any differences in polypeptide composition. From the present data, it is estimated that there are about 4 X 10(9) molecules of RNA polymerase III per oocyte nucleus. This extraordinary level of soluble RNA polymerase III in part explains the ability of oocyte nuclei (or extracts) to support the active transcription of exogenous tRNA and 5 S RNA genes. The present and previous data concerning the properties and structures of the oocyte versus somatic cell enzymes also suggest that the RNA polymerase III present in the oocyte is functionally equivalent to a somatic cell enzyme and support the previous suggestion that this enzyme is conserved and used later in (somatic) embryonic cells.

Animals↗
PubMed1993

Site-specific initiation of transcription by RNA polymerase II.

RNA polymerase II initiates transcription at specific DNA sequences. Studies using sequence analysis and molecular genetics suggest a simple and universal model of start-site selection by RNA polymerase II. Two consensus sequences occur at fixed positions in promoters from higher eukaryotes and their viruses: the TATA box around -30 and the initiator at the start site of transcription. Both consensus sequences function as positioning elements that control site-specific initiation. As a first step during initiation, the basal transcription factor TFIID binds to the TATA box; regulatory transcription factors can tether TFIID bind to the TATA box; regulatory transcription factors can tether TFIID to promoters without a consensus TATA box. TFIID then directs the assembly of other basal transcription factors and RNA polymerase II into a preinitiation complex. Finally, RNA polymerase II searches for the best match to the initiator consensus about 30 base pairs downstream of the TATA box to select the exact start site. The transcriptional activity of a start-site sequence generally correlates with its similarity to the initiator consensus, suggesting that there is only one type of initiator.

Base Sequence↗
PubMed1993

Physical studies on interaction of transcription activator and RNA-polymerase: fluorescent derivatives of CRP and RNA polymerase.

Protein-protein interactions between cAMP receptor protein (CRP) and RNA polymerase (RNAP) have been proposed to be essential in RNAP activation by CRP in type I promoters. These two proteins were shown to interact in solution in the absence of promoter DNA (Heyduk et al., 1993). In this report we describe the preparation of fluorescent derivatives of CRP (fluorescent probes at position 13 and 85); and of the alpha-subunit of RNAP (at position 321). The specific incorporation of fluorescence probes was achieved by expressing protein in a bacteria strain, auxotrophic for tryptophan, in media containing 5-hydroxytryptophan (5-OH-Trp). The absorbance spectrum of a protein containing 5-OH-Trp is shifted towards longer wavelengths as compared to the native protein. This allows selective monitoring of the fluorescence signal of 5-OH-Trp derivative of a protein even in the presence of high concentration of tryptophan containing protein(s). The CRP derivative is shown to retain 100% of the native protein cAMP binding and specific DNA binding activity. Using a fluorescence polarization assay, it is also shown that 5-OH-Trp derivative of CRP interacts with RNAP as well as the native protein. The RNAP reconstituted with 5-OH-Trp derivative of the alpha-subunit retained the enzymatic activity. Fluorescence quenching studies show that Trp 321 of alpha-subunit is located in the region of the protein which is exposed to a solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Hydroxytryptophan↗
PubMed1989

An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains.

We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum. The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3. The P. falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits. A distinctive carboxyl-terminal domain contains a short highly conserved heptapeptide repeat domain which is bounded on its 5' side by a highly diverged heptapeptide repeat domain, and is bounded on its 3' side by a long carboxyl-terminal extension.

Amino Acid Sequence↗
PubMed2003

Positive and negative regulators for neuronal BC1 RNA transcription by RNA polymerase III are possible members of the RNA polymerase II transcription system.

Neuronal cell-specific BC1 RNA is a unique RNA polymerase III (Pol III) transcript. The transcription is controlled by an activator E2 site and by BCRE, a repressor element, in response to neuronal activity. BC1 RNA is localized to dendritic domains as ribonucleoprotein particles, and it has been suggested to play a functional role in translational regulation of dendritic mRNAs. In the present study, using a luciferase assay in NG108-15 cells, we found that the positive and negative regulators for BC1 RNA transcription can also function in the Pol II transcription system. Our results suggest that the neuronal activity-dependent expression of BC1 RNA by Pol III and a subset of neuronal mRNAs by Pol II may be simultaneously controlled by the E2 site and BCRE, as well as their binding proteins.

Animals↗
PubMed1994

From initiation to elongation: comparison of transcription by prokaryotic and eukaryotic RNA polymerases.

Multisubunit RNA polymerases in prokaryotes and eukaryotes share an evolutionarily conserved core. Here, we compare the processes of promoter recognition, transcription initiation and transcript elongation by human RNA polymerase II and by the RNA polymerase of the eubacterium Escherichia coli. Although these two polymerases have diverged widely in structure, important functions have been conserved, suggesting that the basic mechanisms of RNA transcription are similar in eukaryotes and prokaryotes.

DNA-Directed RNA Polymerases↗
PubMed1988

RPA190, the gene coding for the largest subunit of yeast RNA polymerase A.

Yeast RNA polymerases are being extensively studied at the gene level. The entire gene encoding the largest subunit of RNA polymerase A, A190, was isolated and characterized in detail. Southern hybridization and gene disruption experiments showed that the RPA190 gene is unique in the haploid yeast genome and essential for cell viability. Nuclease S1 mapping was used to identify mRNA 5' and 3' termini. RPA190 encodes a polypeptide chain of 186,270 daltons in a large uninterrupted reading frame. A dot matrix comparison of the deduced amino acid sequence of subunit A190 with Escherichia coli beta' and cognate subunits B220 and C160 from yeast RNA polymerases B and C showed a conserved pattern of homology regions (I-VI). A potential DNA-binding site (zinc-binding motif) is conserved in the N-terminal region I. Remarkably, the A190 subunit does not harbor the heptapeptide repeated sequence present in the B220 subunit. The sequence of the A190 subunit diverges from B220 and C160 by the presence of two hydrophilic domains inserted between homology regions I and II, and V and VI. From their codon usage and third base pyrimidine bias, RNA polymerase genes RPA190, RPB220, RPC160, and RPC40 fall among yeast genes expressed at an average level. The RPA190 5'-flanking region contains features present in other polymerase genes that might function in regulation.

Amino Acid Sequence↗
PubMed2005

Structure of an RNA polymerase II-RNA inhibitor complex elucidates transcription regulation by noncoding RNAs.

The noncoding RNA B2 and the RNA aptamer FC bind RNA polymerase (Pol) II and inhibit messenger RNA transcription initiation, but not elongation. We report the crystal structure of FC(*), the central part of FC RNA, bound to Pol II. FC(*) RNA forms a double stem-loop structure in the Pol II active center cleft. B2 RNA may bind similarly, as it competes with FC(*) RNA for Pol II interaction. Both RNA inhibitors apparently prevent the downstream DNA duplex and the template single strand from entering the cleft after DNA melting and thus interfere with open-complex formation. Elongation is not inhibited, as nucleic acids prebound in the cleft would exclude the RNA inhibitors. The structure also indicates that A-form RNA could interact with Pol II similarly to a B-form DNA promoter, as suggested for the bacterial transcription inhibitor 6S RNA.

Base Sequence↗
PubMed1992

In vitro interactions of Pseudomonas RNA polymerases with tac and RNA I promoters.

The activities of RNA polymerases from Pseudomonas putida and Pseudomonas aeruginosa were compared with that of Escherichia coli RNA polymerase in an in vitro transcription system. All three enzymes initiated transcription at the tac promoter and the RNA I promoter of E. coli. We measured the rate of open complex formation between the RNA polymerases and the promoters, and the saturation level of open complex formation at equilibrium in single-round transcription. The relative rates of open complex formation were P. putida > E. coli > P. aeruginosa and the relative saturation levels of open complex formation at equilibrium were E. coli > P. putida > P. aeruginosa for the tac and RNA I promoters. The interaction of the RNA polymerases with the promoters was also studied by DNase I footprinting. The patterns of protection of the Pseudomonas RNA polymerases on the tac promoter were similar to that of E. coli RNA polymerase. However, the protection patterns of the Pseudomonas RNA polymerases on the RNA I promoter were slightly different from that of E. coli RNA polymerase.

DNA Fingerprinting↗
PubMed1995

Gene RPA43 in Saccharomyces cerevisiae encodes an essential subunit of RNA polymerase I.

Yeast RNA polymerase I contains 14 distinct polypeptides, including A43, a component of about 43 kDa. The corresponding gene, RPA43, encodes a 326-amino acid polypeptide matching the peptidic sequence of two tryptic fragments isolated from A43. Gene inactivation leads to a lethal phenotype that is rescued by a plasmid containing the 35S ribosomal RNA gene fused to the GAL7 promoter, which allows the synthesis of 35S rRNA by RNA polymerase II in the presence of galactose. A screening for mutants rescued by the presence of GAL7-35SrDNA identified a nonsense rpa43 allele truncating the protein at amino acid position 217. [3H]Uridine pulse labeling showed that this mutation abolishes 35S rRNA synthesis without significant effects on the synthesis of 5 S RNA and tRNAs. These properties establish that A43 is an essential component of RNA polymerase I. This highly hydrophilic phosphoprotein has a strongly acidic carboxyl-terminal domain, and shows no homology to entries in current sequence data banks, including all the genetically identified components of the other two yeast RNA polymerases. RPA43 mapped next to RPA190, encoding the largest subunit of polymerase I. These genes are divergently transcribed and may thus share upstream regulatory elements ensuring their co-regulation.

Amino Acid Sequence↗