Purified RNA Polymerase Research Articles

The enhanced rate of transcription of methyl mercury-exposed DNA by RNA polymerase is not sufficient to explain the stimulatory effect of methyl mercur

Previous work demonstrated two stimulatory effects of methyl mercury on nucleic acid synthesis: (1) in isolated nuclei, methyl mercury stimulates RNA synthesis which is catalyzed by RNA polymerase II [Frenkel and Randles, J. Biol. Chem. 257, 6275-6279 (1982)]. (2) Brief exposure of purified DNA to methyl mercury increases the rate of its transcription by purified RNA polymerase II [Frenkel, Cain, and Chao, Biochem. Biophys. Res. Commun. 127, 849-856 (1985)]. The latter effect was considered as a possible mechanism of the former. Two lines of evidence are presented here which demonstrate that the latter effect is not a sufficient explanation for the former. (1) Mercuric perchlorate has been found to increase the rate of DNA transcription by purified polymerase and the template properties of the mercuric perchlorate-exposed DNA have been found to resemble those of methyl mercury-exposed DNA. Nevertheless, mercuric perchlorate has been shown not to stimulate RNA synthesis in isolated HeLa nuclei. (2) In isolated nuclei of the B50 rat neuroblastoma cell line, RNA synthesis has been found to be stimulated only minimally by methyl mercury. Nevertheless, RNA polymerase II purified from the B50 cells has been found to transcribe methyl mercury-exposed DNA at a higher rate than unexposed control DNA.

Production of monoclonal antibodies against RNA polymerase I from nonimmunized autoimmune MRL/lpr mice and their use in rDNA transcription analysis.

A hybridoma secreting monoclonal antibodies against RNA polymerase I was produced by the fusion of myeloma cells with spleen cells from a nonimmunized MRL/lpr mouse which is known to produce autoantibodies to RNA polymerase I. The antibodies (McAb-2D11) belong to the IgG2b subclass, reacted specifically with the second largest (120 kDa) subunit of RNA polymerase I, and inhibited accurate transcription of cloned rat rDNA in a fractionated cell extract following immunoprecipitation of RNA polymerase I. McAb-2D11 did not inhibit RNA polymerase II-mediated transcription of the mouse metallothionein-I gene. Immunocytochemical procedures with biotinylated second antibody demonstrated specific immunolocalization of RNA polymerase I in the nucleus. These studies have (a) presented direct evidence that autoantibodies to functional RNA polymerase I are produced in a murine model of systemic lupus erythematosus, (b) demonstrated specificity of the monoclonal antibody for RNA polymerase I, and (c) provided a useful tool for the purification of RNA polymerase I and/or transcription factor(s) associated with RNA polymerase I.

Expression of enzymatically active poliovirus RNA-dependent RNA polymerase in Escherichia coli.

The poliovirus genome is replicated by a virus-encoded RNA-dependent RNA polymerase (RNA polymerase). The RNA polymerase is first synthesized as a larger precursor polypeptide, which is subsequently processed by a viral proteinase, 3Cpro, to give the mature polymerase molecule, 3Dpol. To further characterize the poliovirus RNA polymerase, we have constructed plasmids that expressed this protein in Escherichia coli. The plasmids consisted of fusions between the E. coli DNA encoding the first 13 amino acids of the trp operon leader protein and viral genes encoding the 3Cpro and 3Dpol polypeptides. E. coli harboring such plasmids gave significant, inducible levels of enzymatically active RNA polymerase as determined by the poly(A).oligo(U) poly(U) polymerase assay. The purified RNA polymerase activity from E. coli corresponded to a protein with the approximate molecular weight of the mature 3Dpol protein. The availability of a recombinant, enzymatically active poliovirus RNA polymerase provides a system in which we can precisely delineate the role this enzyme plays in the regulation of poliovirus replication.

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 hydro-cortisone 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 the degree observed in vitro following in vivo hydrocortisone administration.

Identification of intrinsic termination sites in vitro for RNA polymerase II within eukaryotic gene sequences

We have identified and mapped several DNA sequences within a human histone gene (H3.3) at which in-vitro transcription by highly purified RNA polymerase II is efficiently terminated. Since transcription in our system involves only RNA polymerase II acting on a linear DNA template, these sequences contain “intrinsic” termination signals recognized by the polymerase protein itself. The existence of such signals within a gene suggests that efficient antitermination systems probably exist for mammalian transcription units. Alternatively, there could be a high frequency of premature transcription termination, or “polarity” for genes such as H3.3. Intrinsic transcription termination sites in H3.3 are located in sequences of consecutive thymidylate residues (5 to 8 nucleotides) on the non-transcribed DNA strand (T-runs), from which it is likely that such T-runs are elements of the intrinsic termination signal for RNA polymerase II. However, transcription proceeds without significant termination through many similar T-runs, from which it follows that these intrinsic termination signals include other elements. Since transcription is also terminated efficiently at these sites when the transcript remains bound along its full length as a DNA-RNA hybrid, it is unlikely that formation of specific RNA secondary structures in the transcript is a general element of the intrinsic termination signal. Although DNA sequences downstream from the coding portion of the mouse β-globin gene have been implicated as sites of transcription termination in vivo, these regions do not contain strong intrinsic termination signals, and transcription in vitro proceeds through these regions almost undiminished. Transcriptional termination in this region in vivo may depend on the presence of termination factors or other intracellular elements, and there may be multiple classes of DNA signals that control eukaryotic termination.

Purified RNA polymerase II recognizes specific termination sites during transcription in vitro.

We have studied the ability of certain well-defined prokaryotic DNA sequences to act as specific termination signals for highly purified calf thymus RNA polymerase II. We used duplex DNA fragments modified to direct efficient and specific transcription of defined DNA templates to follow transcription with RNA polymerase alone in the absence of additional protein factors. Elongation of RNA chains by RNA polymerase II is processive through most DNA sequences. However, certain DNA sequences serve as effective "intrinsic" terminators for RNA polymerase II; in this they resemble the "rho-independent" terminators for the bacterial RNA polymerase. Several rho-independent bacterial terminators are also able to act as termination signals for RNA polymerase II. However, there is no apparent correlation between the efficiency of termination for the bacterial enzyme and that found for the calf thymus enzyme. One very efficient bacterial terminator (phage T7 early terminator) gives no termination with RNA polymerase II, and we have identified at least two sites that cause the eukaryotic enzyme to terminate but have no effect on transcription by the bacterial enzyme. Hence, the signals recognized as intrinsic termination sites for the two enzymes are substantially different. All of the sites that act as intrinsic terminators for RNA polymerase II contain a series of consecutive thymidine residues in the nontranscribed DNA strand (T-run), and the 3' end of the completed RNA normally lies within this sequence. It is plausible that the T-run is part of the signal for an RNA polymerase II termination site; however, there is no apparent correlation between the number of T residues and the efficiency of the terminator, suggesting that other sequence elements are required for, or modulate, termination. Several lines of evidence suggest that the formation of RNA secondary structures in the nascent transcript is not an essential element of the intrinsic RNA polymerase II termination signal.

Inactivation of the RNA polymerase of vesicular stomatitis virus by N-ethylmaleimide and protection by nucleoside triphosphates. Evidence for a second ATP binding site on L protein.

The purified RNA polymerase complex of vesicular stomatitis virus required added thiols for maximal activity, whereas polymerase activity from whole disrupted virions did not. Maximal activity of the purified polymerase complex required greater than or equal to 1 mM added dithiothreitol. The polymerase was inactivated by N-ethylmaleimide (NEM) at 0 degree C, with k2 = 528 +/- 26 M-1 min-1. Activity was recovered by addition of L protein, but not N or NS, to the NEM-inactivated complex, indicating that the NEM-sensitive group was present on the L protein. Nucleoside triphosphates protected the enzyme against inactivation by N-ethylmaleimide. ATP was most effective, with KD = 0.58 +/- 0.07 mM, a value close to the Km of ATP reported previously for initiation of RNA synthesis. dATP was nearly as effective, and GTP was slightly less effective than ATP. Non-hydrolyzable analogs of ATP protected weakly, whereas ADP and pyrimidine triphosphates gave very poor, but still measurable, protection. The ATP binding site thus identified differs from the protein kinase-associated ATP binding site identified on L protein by Sanchez et al. (Sanchez, A., De, B.P., and Banerjee, A. K. (1985) J. Gen. Virol. 66, 1025-1036) in having a substantially lower affinity for ATP. Two putative ATP binding sites were identified in the L protein amino acid sequence, but none were found in the N or NS sequences.

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.

The 180 KDa polypeptide contains the DNA-binding domain of RNA polymerase II

Purification of RNA polymerase II from chicken myeloblastosis (leukemia) cells to homogeneity and subsequent structural analysis of the purified enzyme revealed that the enzyme contained seven polypeptides with molecular masses ranging from 27 KDa to 220 KDa. Inclusion of protease inhibitors in the buffer system during purification significantly increased the molar ratio of the largest (220 KDa) polypeptide to the second largest (180 KDa) polypeptide. However, proteolytic conversion of the 220 KDa to 180 KDa polypeptide did not inhibit the DNA binding activity of the enzyme. The enzyme, after dissociation into subunits in a SDS-polyacrylamide gel containing urea was blotted onto a nitrocellulose filter. The filter was incubated with 32P-labeled calf thymus DNA and both the 220 KDa and 180 KDa polypeptides of the enzyme bind DNA, suggesting that the DNA-binding site of the enzyme resides on the 180 KDa polypeptide of the largest subunit.

Transcriptional specificity after mycobacteriophage I3 infection

Transcriptional regulation following mycobacteriophage I3 infection was investigated. For this purpose, RNA polymerase mutants (rifr) of the host bacterium, Mycobacterium smegmatis, were isolated and characterized. Phage growth in rifsand rifr cells in the presence of rifampicin revealed the involvement of host RNA polymerase in phage genome transcription. This was confirmed by studies on in vivo RNA synthesis as well as by direct RNA polymerase assay after phage infection. Significant stimulation in RNA polymerase activity was seen following phage infection. The maximal levels were attained in about 60 min post infection and maintained throughout the phage development period. The stimulation of polymerase activity was most pronounced when the phage DNA was used as the template. RNA polymerases from uninfected and phage-infected M. smegmatis were purified to homogeneity. Enzyme purifn. was accomplished by a rapid procedure utilizing affinity chromatog. on rifampicin-Sepharose columns. Subunit structure anal. of the purified RNA polymerase from uninfected and phage-infected cells showed the presence of \alpha, \beta, \beta' and \sigma subunits similar to the other prokaryotic RNA polymerases. In addn., a polypeptide of 79,000 daltons was assocd. with the enzyme after phage infection. The enzymes differed in their properties with respect to template specificity. Phage I3 DNA was the preferred template for the modified RNA polymerase isolated from infected cells, which may account for the transcriptional switch required for phage development.

Factors involved in specific transcription by mammalian RNA polymerase II. Purification and functional analysis of initiation factors IIB and IIE.

Two general transcription factors (IIE and IIB) (TF) were purified from HeLa cell nuclear extracts and shown to be absolutely required, along with two additional factors (IIA and IID) and RNA polymerase II, for specific transcription initiation at the adenovirus major late promoter. TFIIB and TFIIE were also required, in addition to TFIIA, TFIID, RNA polymerase II, and the adenovirus 2 major late promoter, for the formation of a (preinitiation) complex that could initiate transcription (upon addition of nucleoside triphosphates) in the presence of heparin concentrations which inhibited the action of unbound factors. Glycerol gradient analyses indicated independent interactions of TFIIE with TFIIB and with the purified RNA polymerase II, but not with RNA polymerase III. Transcription factors IIB and IIE were also shown to be required for specific initiation of transcription from several cellular and viral class II promoters.

Novobiocin inhibits interactions required for yeast TFIIIB sequestration during stable transcription complex formation in vitro.

Novobiocin concentrations normally used to inhibit a putative eukaryotic DNA gyrase have been found to inhibit transcription of a yeast 5S rRNA gene using an in vitro yeast transcription system. Purified RNA polymerase III and three yeast transcription factors (chromatographically separated, partially purified and free of any detectable gyrase activity) were used. Novobiocin prevents specific transcription if added to the in vitro system immediately prior to the addition of transcription factors and RNA polymerase. If a stable transcription factor complex is allowed to form prior to the addition of novobiocin, concentrations of novobiocin as high as 1000 micrograms/ml have no effect on in vitro transcription. Transcription factors TFIIIA and TFIIIC are able to be stably sequestered onto 5SrDNA-cellulose, but factor TFIIIB is not able to associate with the 5SrDNA-TFIIIA-TFIIIC complex in the presence of novobiocin. Although novobiocin is able to precipitate other basic proteins, it does not appear to precipitate any of these class III gene transcription factors, but instead appears to act by disrupting specific factor-factor interactions.

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 II O, II A, and II B. Although their relative proportions vary among different sources, RNA polymerases II A and II B 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 II O is present in whole cells and isolated nuclei in higher proportions than previously reported. Subspecies II O was found associated with SV40 minichromosomes engaged in transcription during late lytic infection. The observation that RNA polymerase II O is associated with the cellular chromatin and SV40 minichromosomes suggest that this form of the enzyme is the subspecies active in in vivo transcription.

Immunochemical analysis of mammalian RNA polymerase II subspecies. Stability and relative in vivo concentration.

Three subspecies of RNA polymerase II, designated IIO, IIA, and IIB, have been described in a variety of eukaryotic cells and shown to differ in the molecular weight of their largest subunit, designated IIo, IIa, and IIb, respectively. The objectives of this study were to establish the in vivo molecular structure of RNA polymerase II in mammalian cells and to examine conditions that influence the stability of RNA polymerase II subspecies. Subunit affinity-purified antibodies were used to determine the relative concentration of subunits IIo, IIa, and IIb in crude extracts of calf thymus tissue, cultured bovine kidney cells, and HeLa cells. HeLa cells contain exclusively RNA polymerase IIO whereas both cultured bovine kidney cells and calf thymus tissue contain RNA polymerases IIO and IIA. RNA polymerase IIB was not detected at significant levels in any of the cell extracts examined. Cell extracts were aged at either 4 degrees or 37 degrees C and the stability of RNA polymerases IIO and IIA determined by protein blotting. In the presence of buffer normally used for RNA polymerase purification, subunit IIo disappears from calf thymus extracts within 24 h at 4 degrees C or within 5 min at 37 degrees C. RNA polymerase IIO is partially stabilized by the inclusion of protease inhibitors and further stabilized by the presence of relatively high concentration of EDTA and EGTA. The prior fractionation of nuclei does not have an appreciable effect on RNA polymerase II stability. An increase in the amount of reducing agent causes a dramatic reduction in the stability of subunit IIo. The following manuscript (Bartholomew, B., Dahmus, M. E., and Meares, C. F. (1986) J. Biol. Chem. 14226-14231) examines the transcriptional activity of RNA polymerases IIO and IIA in reactions dependent on the major late promoter of adenovirus-2. Photoaffinity labeling of subunits IIo and IIa, relative to their concentration in the transcription extract, indicates that the transcriptional activity of RNA polymerase IIO is greater than 10 times that of IIA.

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. VIII. Localisation of a region involved in promoter selectivity.

We have previously isolated an E. coli derivative carrying a small internal deletion (delta(rpoB)1570-1) of the beta structural gene. This RNA polymerase deletion mutant has no noticeable phenotype other than a slightly increased generation time in minimal medium. The deletion, which removes about 165 bp, has been localised to between codons 965 and 1,083, indicating it excises part of a tandem repeat structure present in the C-terminal region of beta. Analysis in vitro of purified RNA polymerase from the deletion mutant indicates that this enzyme has an altered promoter selectivity. These observations allow localisation of a site on the beta polypeptide of E. coli RNA polymerase involved in transcriptional initiation.

MRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II.

MRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II.

Purification and characterization of a specific RNA polymerase II transcription factor.

Accurate transcription by RNA polymerase II has previously been shown to require a HeLa cell fraction designated [AB] in addition to other components (Samuels, M., Fire, A., and Sharp, P. A. (1982) J. Biol. Chem. 257, 14419-14427). A factor which substituted for HeLa [AB] was identified in chromatographic fractions from calf thymus and was purified an estimated 9,000-fold or more. The final calf thymus [AB] fractions contained three polypeptide species with molecular weights of 19,600, 19,100, and 12,800, whose appearance correlated with the transcription factor enzymatic activity. The intact factor had a molecular weight of 25,600-35,000 based on gel filtration and sedimentation analysis and could be inactivated by treatment with high temperatures or with N-ethyl-maleimide. [AB] did not stimulate nonspecific nucleotide incorporation by pure RNA polymerase II, but it did interact with factor [DB] and promoter template DNA to form a functional intermediate preceding accurate initiation. The calf thymus factor thus was homologous to HeLa [AB] by both physical and functional criteria. In contrast to a previous suggestion that this factor has properties associated with actin the highly purified active fractions did not contain detectable actin.

Specific in vitro transcription of 16S rRNA gene by pea chloroplast RNA polymerase

A highly purified RNA polymerase preparation from pea chloroplasts has been shown to specifically transcribe the 16S rRNA gene in vitro using the recombinant pCB2-8 DNA as a template. The RNA polymerase has been found to show maximum activity and specificity with pea supercoiled rDNA as a template. At low concentrations of ribonucleoside triphosphates, the RNA polymerase selectively initiates transcription on the 16S rRNA gene. A part of the 16S rRNA gene has been sequenced. The mature 16S rRNA has been found by S1 nuclease analysis to contain sequences starting from GAAGCT. The in vitro synthesized RNA has been found to protect the same nucleotides GAAGCT. In addition, the in vitro synthesized RNA was also found to strongly protect bases starting with TATG located at about 260 bases away from the start site of the mature 16S rRNA.

Evidence for an indirect mechanism of aflatoxin B1 inhibition of rat liver nuclear RNA polymerase II activity in vivo

Previous studies suggested multiple sites of action of aflatoxin B1 (AFB1) in vivo to inhibit rat liver nuclear RNA synthesis--it impairs nucleolar DNA template function and inhibits RNA polymerase II activity. We have previously shown that AFB1 activated in vitro inhibits nucleolar RNA synthesis. The question is whether AFB1 can inhibit RNA polymerase II under these in vitro conditions. Male Sprague-Dawley rats, 200 g, were injected i.p. with 0.6 mg AFB1 and liver nuclei were isolated 2 h later. When the total nuclear free RNA polymerases were extracted and assayed in the absence and presence of alpha-amanitin (3.2 micrograms/ml), we found that only alpha-amanitin-sensitive (i.e., RNA polymerase II) activity was inhibited (97%). DEAE-Sephadex chromatography confirmed this result. When total nuclear free RNA polymerases were incubated with AFB1 activated in vitro under conditions producing 70% inhibition of nucleolar RNA synthesis, no inhibition was observed for either alpha-amanitin-sensitive or -resistant activities. Similar results were obtained with low and high (28 and 167 micrograms/ml) concentrations of AFB1. This was further confirmed using highly purified RNA polymerase II. We conclude that AFB1 inhibition of RNA polymerase II activity in vivo is not a result of direct interaction of AFB1 to the enzyme.