Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA polymerase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed2004

Malondialdehyde adducts in DNA arrest transcription by T7 RNA polymerase and mammalian RNA polymerase II.

Malondialdehyde, a genotoxic byproduct of lipid peroxidation, reacts with guanine in DNA to form pyrimido[1,2-alpha]purin-10(3H)one (M(1)dG), the first endogenous DNA lesion found to be a target of nucleotide excision repair enzymes. A subpathway of nucleotide excision repair, transcription-coupled repair, is thought to occur when RNA polymerase (RNAP) is arrested at damage in transcribed DNA strands and might function for efficient removal of M(1)dG in active genes. Results presented here show that M(1)dG and its stable, exocyclic analog 1,N(2)-propanodeoxyguanine (PdG), arrest translocation of T7 RNAP and mammalian RNAPII when located in the transcribed strand of a DNA template. M(1)dG paired with thymine is exocyclic and poses a stronger block to transcription than the acyclic N(2)-(3-oxo-1-propenyl)-dG, formed upon cytosine-catalyzed opening of M(1)dG in duplex DNA. PdG is a complete block to RNAPII regardless of base pairing. The elongation factor TFIIS (SII) induces reversal and RNA transcript cleavage by RNAPII arrested at PdG. Thus, arrested RNAPII complexes may be stable at M(1)dG in cells and may resume transcription once the offending adduct is removed. The conclusion from this work is that malondialdehyde adducts in the transcribed strand of expressed genes are strong blocks to RNAPs and are targets for cellular transcription-coupled repair. If so, then M(1)dG, already known to be highly mutagenic in human cells, also may contribute to apoptosis in the developing tissues of individuals with Cockayne's syndrome, a hereditary disorder characterized by transcription-coupled repair deficiency.

Animals↗
PubMed2001

Bacterial RNA polymerase subunit omega and eukaryotic RNA polymerase subunit RPB6 are sequence, structural, and functional homologs and promote RNA polymerase assembly.

Bacterial DNA-dependent RNA polymerase (RNAP) has subunit composition beta'betaalpha(I)alpha(II)omega. The role of omega has been unclear. We show that omega is homologous in sequence and structure to RPB6, an essential subunit shared in eukaryotic RNAP I, II, and III. In Escherichia coli, overproduction of omega suppresses the assembly defect caused by substitution of residue 1362 of the largest subunit of RNAP, beta'. In yeast, overproduction of RPB6 suppresses the assembly defect caused by the equivalent substitution in the largest subunit of RNAP II, RPB1. High-resolution structural analysis of the omega-beta' interface in bacterial RNAP, and comparison with the RPB6-RPB1 interface in yeast RNAP II, confirms the structural relationship and suggests a "latching" mechanism for the role of omega and RPB6 in promoting RNAP assembly.

Amino Acid Sequence↗
PubMed1998

Recognition of a human arrest site is conserved between RNA polymerase II and prokaryotic RNA polymerases.

DNA sequences that arrest transcription by either eukaryotic RNA polymerase II or Escherichia coli RNA polymerase have been identified previously. Elongation factors SII and GreB are RNA polymerase-binding proteins that enable readthrough of arrest sites by these enzymes, respectively. This functional similarity has led to general models of elongation applicable to both eukaryotic and prokaryotic enzymes. Here we have transcribed with phage and bacterial RNA polymerases, a human DNA sequence previously defined as an arrest site for RNA polymerase II. The phage and bacterial enzymes both respond efficiently to the arrest signal in vitro at limiting levels of nucleoside triphosphates. The E. coli polymerase remains in a template-engaged complex for many hours, can be isolated, and is potentially active. The enzyme displays a relatively slow first-order loss of elongation competence as it dwells at the arrest site. Bacterial RNA polymerase arrested at the human site is reactivated by GreB in the same way that RNA polymerase II arrested at this site is stimulated by SII. Very efficient readthrough can be achieved by phage, bacterial, and eukaryotic RNA polymerases in the absence of elongation factors if 5-Br-UTP is substituted for UTP. These findings provide additional and direct evidence for functional similarity between prokaryotic and eukaryotic transcription elongation and readthrough mechanisms.

Base Sequence↗
PubMed1978

Role of the 21,000 molecular weight polypeptide of Bacillus subtilis RNA polymerase in RNA synthesis.

DNA-dependent RNA polymerase from Bacillus subtilis contains a 21,000 molecular weight (21K) peptide subunit. This subunit was purified and added to 21K-depleted polymerase isolated from both uninfected and SP85-infected B. subtilis. The effect of the subunit on total RNA synthesis, on enzyme-DNA binding, on RNA chain initiation and elongation, and on enzymatic specificity were examined. A comparison was made of the effects of the 21K peptide and NaCl on polymerase activity, RNA chain elongation, and symmetry of transcription of SP82 DNA. The addition of the 21K peptide to enzyme preparations lacking this subunit stimulated total polymerase activity 20 to 40%. In contrast, addition of NaCl at concentrations greater than 0.1 M significantly reduced polymerase activity. The 21K peptide appeared to alter the general affinity of the polymerase for DNA. The rate of RNA chain initiation was not affected by the 21K peptide, but RNA chain elongation was stimulated. Both the 21K peptide and NaCl increased the asymmetry of transcription of SP82 DNA by phage-modified polymerase. The 21K effect was related to the stimulation of elongation while high concentrations of NCl appeared to act at RNA chain initiation. RNAs synthesized in vitro by polymerase lacking and supplemented with the 21K peptide were translated by a Escherichia coli cell-free system. The 21K peptide had little direct effect on the selection of promoters in vitro as measured by this technique, but it dramatically increased the translatability of the product.

Bacillus subtilis↗
PubMed1986

Structure of monkey kidney cell RNA polymerase II: characterization of RNA polymerase associated with SV40 late transcriptional complexes.

Three subspecies of RNA polymerase II have been described in eucaryotic cells and designated IIO, IIA, and IIB. Although their relative proportions vary among different sources, RNA polymerases IIA and IIB constitute the bulk of most purified RNA polymerase II preparations. Antibodies against calf thymus RNA polymerase II were used to estimate the amount of polymerase II subspecies in monkey kidney cells, isolated nuclei, and SV40 late transcriptional complexes. We have found that RNA polymerase IIO is present in whole cells and isolated nuclei in higher proportions than previously reported. Subspecies IIO was found associated with SV40 minichromosomes engaged in transcription during late lytic infection. The observation that RNA polymerase IIO is associated with the cellular chromatin and SV40 minichromosomes suggest that this form of the enzyme is the subspecies active in in vivo transcription.

Animals↗
PubMed1996

Onset of C. elegans gastrulation is blocked by inhibition of embryonic transcription with an RNA polymerase antisense RNA.

Cleavage and gastrulation initiation in Caenorhabditis elegans embryos are characterized by an invariant temporal and spatial pattern of cell divisions and cell movements. Although bulk embryonic transcription does not begin until gastrulation onset, some transcription can be detected as early as the 4-cell stage. To determine whether any early transcripts are required for normal cleavage-stage patterning, we blocked transcription in embryos by injecting hermaphrodite parental gonads with RNA antisense to the ama-1 gene, which encodes the large subunit of RNA polymerase II. This treatment prevented the expression of a reporter gene driven by an early embryonic promoter but did not detectably perturb the maternally controlled segregation of the germ line P granules or the pattern of cell division through the first four cleavages. In the fifth cell cycle, however, the two endodermal precursor (E) cells divided early and abnormally and failed to initiate gastrulation. The embryos arrested between the sixth and seventh cell cycles with less than 100 cells. These results indicate that embryonically transcribed gene products are required for gastrulation initiation. They also demonstrate the efficacy of a method for blocking embryonic transcription that may be useful in other organisms.

Animals↗
PubMed1981

Comparison of in vitro chromatin transcription using E. coli RNA polymerase and wheat germ RNA polymerase B.

Use of Escherichia coli RNA polymerase for in vitro transcription of chromatin results in the formation of double-stranded RNA molecules, which consist of a strand of endogenous mRNA and a complementary strand of de novo synthesized RNA. Unless the duplex structures are dissociated prior to isolation of the in vitro transcripts on sulfhydryl agarose columns, the endogenous mRNA can result in over-estimates of in vitro gene-specific transcription. Substitution of wheat germ RNA polymerase B for the bacterial enzyme overcomes this artifact. When mouse fetal liver chromatin is used as template, most of the mRNA synthesized by the plant enzyme is in a single-stranded form. More importantly, this synthesis is directed by a DNA template. Hybridization studies suggest that in vitro transcription of chromatin with wheat germ RNA polymerase B maintains some fidelity to genetic restrictions which operate in vivo.

Animals↗
PubMed1998

The LEF-4 subunit of baculovirus RNA polymerase has RNA 5'-triphosphatase and ATPase activities.

The baculovirus Autographa californica nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that is required for transcription of viral late genes. This polymerase is composed of four equimolar subunits, LEF-8, LEF-4, LEF-9, and p47. The LEF-4 subunit has guanylyltransferase activity, suggesting that baculoviruses may encode a full complement of capping enzymes. Here we show that LEF-4 is a bifunctional enzyme that hydrolyzes the gamma phosphates of triphosphate-terminated RNA and also hydrolyzes ATP and GTP to the respective diphosphate forms. Alanine substitution of five residues previously shown to be essential for vaccinia virus RNA triphosphatase activity inactivated the triphosphatase component of LEF-4 but not the guanylyltransferase domain. Conversely, mutation of the invariant lysine in the guanylyltransferase domain abolished the guanylyltransferase activity without affecting triphosphatase function. We also investigated the effects of substituting phenylalanine for leucine at position 105, a mutation that results in a virus that is temperature sensitive for late gene expression. We found that this mutation had no significant effect on the ATPase or guanylyltransferase activity of LEF-4 but resulted in a modest decrease in RNA triphosphatase activity.

Acid Anhydride Hydrolases↗
PubMed1988

Two forms of RPO41-dependent RNA polymerase. Regulation of the RNA polymerase by glucose repression may control yeast mitochondrial gene expression.

We have identified two chromatographically separable forms of mitochondrial RNA polymerase from Saccharomyces cerevisiae which utilize different DNA templates. One form is only active in a nonselective assay utilizing a poly[d(A-T)] template. The other form selectively initiates from a mitochondrial promoter consensus sequence. Both enzymes can be extracted from yeast mitochondria and all components are encoded by nuclear genes. The possibility that these two activities represent core and holoenzyme forms of the multicomponent mitochondrial RNA polymerase is supported by our observation that both enzymes are absent from a strain bearing a disrupted copy of the RPO41 gene (Greenleaf, A. L., Kelly, J. L., and Lehman, I. R. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3391-3399). The two enzyme activities are differentially regulated by carbon source; the nonselective enzyme is repressed during growth on glucose relative to the selective enzyme. The 5-fold increase in RNA polymerase activity on a nonrepressing carbon source correlates with the increased level of transcript production from mitochondrial DNA. These results suggest that the mitochondrial RNA polymerase and, in consequence, mitochondrial transcription are regulated by carbon catabolite control.

Chromatography↗
PubMed1987

Studies of the functional topography of Escherichia coli RNA polymerase. Affinity labelling of RNA polymerase in a promoter complex by phosphorylating derivatives of primer oligonucleotides.

Amidation of the 5'-phosphate group of the heptanucleotide pdApdApdApdTpdCpdGprC and of its derivatives of the general formula (pdN)npdGprC (n = 0-5) with imidazole, N-methylimidazole, and 4-dimethylaminopyridine afforded a series of phosphorylating affinity reagents. The parent oligonucleotides of this series complementary to promoter A2 of T7 phage over the region (-5 to +2) are known to be efficient primers of the synthesis of RNA by Escherichia coli RNA polymerase with promoter A2 as template. Treatment of the complex RNA-polymerase X promoter-A2 with affinity reagents followed by addition of [alpha-32P]UTP resulted in labelling of RNA polymerase by the residues -(pdN)npdGprCprU (p = radioactive phosphate). This affinity labelling was highly selective because elongation of the covalently bound residues (pdN)npdGprC by prU residues was catalyzed by the active center of RNA polymerase. The most efficient reagents were N-methylimidazolides. A dramatic change of the pattern of labelling of the subunits beta, beta', and sigma took place with changing n. Maximum labelling of the beta subunit occurred at n = 1 and of the sigma subunit at n = 5. The targets in both the subunits were His residues. The alpha subunit was not specifically labelled.

Affinity Labels↗
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↗
PubMed2001

Identification and characterization of a Plasmodium falciparum RNA polymerase gene with similarity to mitochondrial RNA polymerases.

Nearly all mitochondrial RNA polymerase genes identified to date are encoded in the nucleus and have similarities to T3 and T7 bacteriophage RNA polymerases. Some chloroplast genes are also transcribed by T3/T7 phage-like RNA polymerases, raising the possibility that the apicomplexan parasites, which have both a mitochondrion and a plastid, might have two such genes. As part of an investigation of Plasmodium falciparum organelle transcription, we initiated a search for T3/T7 bacteriophage-like RNA polymerase genes. We employed degenerate primers based on highly conserved plant, animal and fungal mitochondrial RNA polymerase sequences to amplify corresponding P. falciparum sequences by polymerase chain reaction (PCR). Less well-conserved flanking sequences were obtained by inverse PCR. The resulting sequence predicts a 1503 amino acid open reading frame with similarity to other T3/T7 phage-like RNA polymerases. Essential amino acids that have been identified in T7 mutant analyses are conserved in the P. falciparum RNA polymerase gene. Comparison of the sequence with preliminary data from the P. falciparum genome sequencing project revealed strain heterogeneity within two regions of the gene. The amino-terminal predicted amino acid sequence of the RNA polymerase gene has similarities to mitochondrial targeting sequences. Taken together, these points suggest that we have identified the P. falciparum mitochondrial RNA polymerase gene.

Amino Acid Sequence↗
PubMed1991

The cloned RNA polymerase II transcription factor IID selects RNA polymerase III to transcribe the human U6 gene in vitro.

Although the human U2 and U6 snRNA genes are transcribed by different RNA polymerases (i.e., RNA polymerases II and III, respectively), their promoters are very similar in structure. Both contain a proximal sequence element (PSE) and an octamer motif-containing enhancer, and these elements are interchangeable between the two promoters. The RNA polymerase III specificity of the U6 promoter is conferred by a single A/T-rich element located around position -25. Mutation of the A/T-rich region converts the U6 promoter into an RNA polymerase II promoter, whereas insertion of the A/T-rich region into the U2 promoter converts that promoter into an RNA polymerase III promoter. We show that this A/T-rich element can be replaced by a number of TATA boxes derived from mRNA promoters transcribed by RNA polymerase II with little effect on RNA polymerase III transcription. Furthermore, the cloned RNA polymerase II transcription factor TFIID both binds to the U6 A/T-rich region and directs accurate RNA polymerase III transcription in vitro. Mutations in the U6 A/T-rich region that convert the U6 promoter into an RNA polymerase II promoter also abolish TFIID binding. Together, these observations suggest that in the human snRNA promoters, unlike in mRNA promoters, binding of TFIID directs the assembly of RNA polymerase III transcription complexes, whereas the lack of TFIID binding results in the assembly of RNA polymerase II snRNA transcription complexes.

Base Sequence↗
PubMedDate not supplied

[Biological properties of Agrobacterium tumefaciens RNA-polymerase. I. Isolation of RNA-polymerase from Agrobacterium tumefaciens 8628].

RNA-polymerase is isolated from Agrobacterium tumefaciens 8628. When fractioning the protein extract by DEAE-cellulose, RNA-polymerase is separated into three fractions eluated with 0.15, 0.165, and 0.185 M KCI. Their electrophoresis in polyacrylamide gel shows that the composition of subunits in the second and third fractions is, in principle, similar to that of Escherichia coli RNA-polymerase, but the first fraction protein distribution pattern differs from he mentioned fractions. The data obtained may evidence for the presence of several RNA-polymerases forms in virulent strains of agrobacteria.

Chromatography, DEAE-Cellulose↗
PubMed1971

RNA polymerases of maize: nuclear RNA polymerases.

Two DNA-dependent RNA polymerases of nuclear origin have been purified from leaves of Zea mays. The two enzymes can be separated on DEAE-cellulose columns. Enzymes I and II are eluted with 0.08 and 0.20 M (NH(4))(2)SO(4), respectively. Both enzymes prefer maize nuclear DNA as a template; they are also more active in the presence of Mg(++) than Mn(++) and are inhibited by (NH(4))(2)-SO(4) or KCl. Neither enzyme is inhibited by rifamycin SV. Enzyme II is strongly inhibited by alpha-amanitin, whereas enzyme I is not significantly affected. Their ability to use native and denatured DNA as templates varies according to the extent and method of purification of the polymerase. Furthermore, enzyme II can be resolved by DEAE-chromatography or glycerol-gradient centrifugation into two components, one of which prefers native DNA, while the other prefers denatured DNA.

Ammonium Sulfate↗