Polymerase Preparations Research Articles

Chloroplast DNA replication in vitro: site-specific initiation from preferred templates.

An enzyme system prepared from maize chloroplasts catalyzes the synthesis of DNA from maize chloroplast DNA sequences cloned in bacterial plasmids. Cloned maize chloroplast DNA fragments Bam HI 17' (2470 bp) and Eco RI x (1368 bp) have been shown to be preferred templates for in vitro DNA synthesis catalyzed by pea chloroplast DNA polymerase preparations [Gold et al. (1987) Proc. Natl. Acad. Sci. USA 84, 194-198]. Analysis of replicative intermediates indicates that although the template activity of the recombinant plasmid pZmcBam 17' is substantially greater than that of the pZmcEco x, replication in both cases originates from within a 455 bp region which overlaps the two plasmids. The remaining approximately 1500 basepair portion of maize chloroplast BamHI fragment 17' is not more active because it contains additional origins for replication. The overlapping region shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that contains a replication origin. Replication is shown to proceed bidirectionally within the 455 bp origin region. Recombinant plasmid pZmc 427, which is also active in the in vitro DNA synthesis assay, promoted localized replication initiation within a 1 kbp Bg1II-Eco RI fragment of the chloroplast DNA insert, a region that includes the 3' terminal part of the psbA gene.

In vitro evidence that the coat protein is the programming factor in alfalfa mosaic virus-induced RNA synthesis

Viral RNA-dependent RNA polymerase preparations were isolated from cowpea mesophyll protoplasts 26 h after inoculation with the complete genome (RNAs 1, 2 and 3) and the incomplete genome (RNAs 1 and 2) of alfalfa mosaic virus. Although in incompletely inoculated protoplasts the ratio minus-/plus-strand RNA synthesis is much higher than in completely inoculated ones [(1983) Virology 124, 75–85)], both enzyme preparations produced largely plus-strand RNA on their endogenous templates. When made template-dependent both were strongly inhibited by small amounts of coat protein. The experiments show that among the two proteins encoded by RNA 3 only the (parental) coat protein can be responsible for the programming of the viral RNA polymerase to plus-strand synthesis.

Transcription of mouse rDNA is regulated by an activated subform of RNA polymerase I

We have identified the species-nonspecific factor required for mouse rDNA transcription, factor C, as an activated subform of RNA polymerase I. C is an RNA polymerase I since it copurifies with bulk polymerase I activity on the three chromatographic columns used to achieve a virtually homogenous preparation of polymerase I, as well as on four additional matrices; it is quantitatively neutralized as well as immunoprecipitated by two different types of anti-polymerase I antibodies; and it has thermal lability identical to that of bulk polymerase I. However, C is clearly distinct from bulk polymerase I in its ability to participate in the stable rDNA transcription complex and to catalyze accurate initiation of rRNA synthesis. It also has a greater sedimentation coefficient than bulk polymerase I. Furthermore, this activated polymerase subform is specifically lacking in extracts of cells in which rDNA transcription was down-regulated because of cycloheximide treatment or attainment of stationary phase. These data suggest that regulation of rDNA transcription in vivo may involve modulation in availability of the activated polymerase I subform.

Transcription analysis of the maize chloroplast gene for the ribosomal protein S4.

Maize seedlings contain several RNA species complementary to the rpS4 coding strand of the maize chloroplast ribosomal protein gene rpS4. All of these have the same 5' end about 182 bp upstream of the translation start codon for the protein S4. Northern and S1 nuclease analyses of RNA isolated from seedlings at different stages of greening show that the size of the pool of rpS4 transcripts does not change significantly upon illumination of dark-grown seedlings. The rpS4 gene has also been analyzed by in vitro transcription using maize chloroplast RNA polymerase preparations. The site of initiation in vitro has been mapped by S1 nuclease analysis to the same location as the 5' terminus of in vivo transcripts. A sequence resembling other plastid promoters occurs just upstream of this initiation site. The sensitivity of in vitro transcription to DNA template superhelicity has been assessed for the rpS4 gene promoter; its negative superhelicity-transcription rate profile resembles that of rbcL.

Yeast mitochondrial RNA polymerase. Purification and properties of the catalytic subunit.

An RNA polymerase was purified 6500-fold to near homogeneity from a whole cell extract of Saccharomyces cerevisiae. The purified enzyme consists of a single 145,000-dalton polypeptide. By subcellular fractionation, the enzyme was localized to the mitochondria. The RNA polymerase activity is alpha-amanitin- and rifampicin-resistant. With single-stranded DNA templates, the enzyme catalyzes the synthesis of polyribonucleotide chains with lengths ranging from less than 10 to greater than 100 residues. It is inactive with double-stranded DNA. Specific antisera inhibit the RNA polymerase activity and recognize the 145,000-dalton polypeptide. The antisera relate this enzyme to previously described yeast mitochondrial RNA polymerase preparations capable of initiation of transcription at mitochondrial promoter sequences (Winkley, C. S., Keller, M. J., and Jaehning, J. A. (1985) J. Biol. Chem. 260, 14214-14223). It therefore appears that the enzyme is a subunit of the yeast mitochondrial RNA polymerase.

Accurate in vitro transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters.

The mitochondrial RNA polymerase from Xenopus laevis oocytes was partially purified by heparin-Sepharose chromatography and phosphocellulose chromatography. This RNA polymerase preparation specifically initiated the transcription of X. laevis mitochondrial DNA (mtDNA) from two bidirectional promoters contained within a 123-base-pair segment of the mtDNA between the heavy-strand replication origin and the rRNA cistrons. Transcription in vitro initiated from precisely the same start sites previously mapped as initiation sites for transcription in vivo. At each of the four sites, initiation occurred within a conserved nucleotide sequence, ACPuTTATA. This consensus sequence is not related to promoters for transcription of human mtDNA.

Accurate in Vitro Transcription of Xenopus laevis Mitochondrial DNA from Two Bidirectional Promoters

The mitochondrial RNA polymerase from Xenopus laevis oocytes was partially purified by heparin-Sepharose chromatography and phosphocellulose chromatography. This RNA polymerase preparation specifically initiated the transcription of X. laevis mitochondrial DNA (mtDNA) from two bidirectional promoters contained within a 123-base-pair segment of the mtDNA between the heavy-strand replication origin and the rRNA cistrons. Transcription in vitro initiated from precisely the same start sites previously mapped as initiation sites for transcription in vivo. At each of the four sites, initiation occurred within a conserved nucleotide sequence, ACPuTTATA. This consensus sequence is not related to promoters for transcription of human mtDNA.

Characterization of the promoter of the large ribosomal RNA gene in yeast mitochondria and separation of mitochondrial RNA polymerase into two different functional components.

We have characterized a DNA sequence that functions in recognition of the promoter of the mitochondrial large rRNA gene by the yeast mtRNA polymerase. Promoter-containing DNA fragments were mutagenized and used as templates to study initiation of transcription in vitro with a partially purified mtRNA polymerase preparation. Deletion mutants, in which increasing stretches of DNA were removed from regions flanking the promoter, define a short area essential for correct initiation of transcription. It virtually coincides with a highly conserved stretch of nine nucleotides that is found immediately upstream of all transcriptional start sites described thus far. Two different point mutations within this nonanucleotide sequence drastically reduce promoter function. Conversely a single point mutation that results in the formation of a nonanucleotide sequence 99 nucleotides upstream of the large rRNA gene leads to a new, efficient transcription initiation site. MtRNA polymerase can be resolved into two different components by chromatography on Blue Sepharose: one retaining the capacity to synthesize RNA, the other conferring the correct specificity of initiation to the catalytic component.

Detection of a subunit of cellular Pol II within highly purified preparations of RNA polymerase isolated from rabbit poxvirus virions

A large (∼200 kd) subunit of cellular RNA polymerase II (Pol II) and a virus-encoded subunit of rabbit poxvirus (RPV) RNA polymerase (137 kd) react with common monoclonal antibodies. Hybridization studies with viral and cellular DNA clones confirm that the viral and cellular proteins are related. Following RPV infection, the Pol II subunit is translocated from the nucleus to the cytoplasmic virosomes, then packaged into mature virus, and is found associated with the viral RNA polymerase purified from virions. The results suggest that the cellular Pol II subunit may be directly involved in the transcription of viral genes.

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.

Sequence and mapping analyses of the herpes simplex virus DNA polymerase gene predict a C-terminal substrate binding domain.

The herpes simplex virus DNA polymerase provides an excellent model for studies of eukaryotic replicative polymerases. We report here the nucleotide sequence of the gene which encodes this enzyme. The gene includes a 3705-base-pair major open reading frame capable of encoding a Mr 136,519 polypeptide, in rough agreement with previous estimates of the size of the major polypeptide found in partially purified viral polymerase preparations. The predicted polymerase polypeptide shares extensive sequence homology with the Epstein-Barr virus open frame predicted to encode DNA polymerase and with a 13-amino acid segment of adenovirus 2 DNA polymerase. Mutations conferring altered sensitivity to antiviral deoxynucleoside triphosphate analogs, pyrophosphate analogs, or aphidicolin from eight different mutants map within the region encoding the carboxyl-terminal portion of the predicted polymerase polypeptide. Two of these are separated by a distance corresponding to at least 228 amino acids. We propose that this region of the gene encodes a polypeptide domain that contains the binding sites for deoxynucleoside triphosphates and pyrophosphate.

Non-disruptive detection of DNA polymerases in nondenaturing polyacrylamide gels.

A non-disruptive method is described with which DNA polymerases can be detected in homogeneous preparations and unfractionated cell extracts after electrophoresis in nondenaturing polyacrylamide gradient gels. The technique involves diffusion of DNA polymerase activity into an overlay assay agarose gel, the synthesis of radioactive DNA, removal of excess substrates and autoradiography. Cell extracts from a variety of organisms were studied using this method. The activity from Escherichia coli crude extracts migrated in a position corresponding to a higher molecular mass than did purified preparations of DNA polymerase I. DNA polymerases of higher organisms generally migrated in positions corresponding to 400--900 kDa, in some cases, close to 200 kDA.

Novel method for identifying sequence-specific DNA-binding proteins.

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.

Novel method for identifying sequence-specific DNA-binding proteins.

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.

Stimulation of the UvrABC enzyme-catalyzed repair reactions by the UvrD protein (DNA helicase II).

An in vitro assay system was constructed using highly purified preparations of UvrA, UvrB, UvrC, UvrD proteins and DNA polymerase I, the objective being to analyse the role of UvrD protein in excision repair of UV-induced DNA damage. UvrABC enzyme-initiated repair synthesis was greatly enhanced by the addition of UvrD protein to the reaction mixture. Further analysis revealed that UvrD protein stimulated introduction of strand breaks in irradiated DNA by UvrABC enzyme but had no effect on the DNA polymerase I reaction. Thus, the site of action of UvrD protein is probably at the incision-excision step and not in later steps in excision repair.

In vitro transcription of herpes simplex virus genes: identification of a new initiation site and second intervening sequence in the immediate-early RNA-5 gene.

We have used partially purified preparations of RNA polymerase II from mock-infected and herpes simplex virus type 1-infected HEp-2 cells to transcribe the herpes simplex virus type 1 strain F BamHI Z DNA fragment containing the promoter for immediate-early RNA-5 (alpha 47), a 1.8-kilobase, spliced RNA. In agreement with the in vivo transcriptional regulation of herpes simplex virus type 1 immediate-early genes, electrophoretic analyses of runoff and truncated transcripts from this template showed that RNA polymerase II from mock-infected cells initiates transcription more selectively than does that from the herpes virus-infected cells at the immediate-early RNA-5 promoter. S1 nuclease mapping of the 5' ends of in vivo- and in vitro-synthesized mRNA placed the initiation site ca. 110 base pairs upstream from the previously published site and also demonstrated the presence of a second, smaller intervening sequence between this new cap site and the previously characterized intervening sequence. S1 analyses also suggested that some splicing of the larger but not the smaller intervening sequence occurred in vitro.

Identification of the yeast DNA polymerase I gene with antibody probes.

Partially overlapping fragments of the gene encoding yeast DNA polymerase I have been cloned by immunological screening of a yeast genomic library constructed in the phage lambda expression vector lambda gt11. The three gene fragments we analyzed in detail encode part of a yeast protein that has been identified as yeast DNA polymerase I, because it shares with this enzyme a number of antigenic determinants. In fact, the yeast protein fragments expressed by the recombinant phages react with both polyclonal and monoclonal antibodies raised against different, highly purified preparations of DNA polymerase I. Moreover, they can be used to affinity purify antibodies specifically reacting with active DNA polymerase I polypeptides and they compete with the yeast enzyme for binding to antibodies that inhibit catalytic activity. The gene is located on chromosome XIV in the yeast genome, and it is transcribed as a 5.2 kb mRNA.

Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (-)-sense genomic RNA.

The genomes of many (+)-stranded RNA viruses, including plant viruses and alphaviruses, consist of polycistronic RNAs whose internal genes are expressed via subgenomic messenger RNAs. The mechanism(s) by which these subgenomic mRNAs arise are poorly understood. Based on indirect evidence, three models have been proposed: (1) internal initiation by the replicase on the (-)-strand of genomic RNA, (2) premature termination during (-)-strand synthesis, followed by independent replication of the subgenomic RNA and (3) processing by nuclease cleavage of genome-length RNA. Using an RNA-dependent RNA polymerase (replicase) preparation from barley leaves infected with brome mosaic virus (BMV) to synthesize the viral subgenomic RNA in vitro, we now provide evidence that subgenomic RNA arises by internal initiation on the (-)-strand of genomic RNA. We believe that this also represents the first in vitro demonstration of a replicase from a eukaryotic (+)-stranded RNA virus capable of initiating synthesis of (+)-sense RNA.

The effect of a bacteriophage T4-induced polypeptide on host RNA polymerase interaction with promoters.

After infection of Escherichia coli with bacteriophage T4, the host RNA polymerase acquires several small phage-induced polypeptides (Stevens, A. (1974) Biochemistry 13, 493-503) and its alpha subunits get ADP-ribosylated by a virus-specific enzyme (Zillig, W., Mailhammer, R., Skorko, R., and Rohrer, H. (1977) Curr. Top. Cell. Regul. 12, 263-271). The modified polymerase displays changed enzymatic properties including sensitivity to increased salt concentration and a higher transition temperature of open promoter complex formation (promoter melting temperature). In order to assess the role of individual modifications in the changed enzyme properties, we isolated RNA polymerase from cells infected with T4 mutant defective in the ADP-ribosylating enzyme. We also purified one of the associated polypeptides, the 15,000-dalton protein which is invariably present in stoichiometric amounts in different RNA polymerase preparations. In an in vitro transcription system using T4 DNA as template, we demonstrate that the 15-kDa protein is the cause of the elevated promoter melting temperature and can induce this property when added to host RNA polymerase. We also show that the increased salt sensitivity of T4-modified polymerase is primarily the result of ADP-ribosylation of its alpha subunits.

Stimulating actions on ribonucleic acid biosynthesis of aconitines, diterpenic alkaloids of Aconitum roots

Mesaconitine (MA) significantly stimulated the incorporation of 5-[ 3H]orotic acid into liver nuclear RNA 16h after its administration. MA exhibited the strongest activity among the aconitine alkaloids. This stimulatory effect of MA was inhibited by actinomycin D, but the incorporation ratio of 5-[ 3H]orotic acid in the combined treatment group with MA and actinomycin D was significantly higher than that in the single treatment group with actinomycin D. MA also increased the incorporation of 5-[ 3H]orotic acid into polysomal RNA in mouse liver. When MA was added to the RNA polymerase (EC 2.7.7.6) preparation obtained from rat liver, the increase of the enzyme activity was weak. While RNA polymerase preparation from the liver of rats, which were previously treated with MA, elicited increased incorporation of [ 3H]cytidine monophosphate into RNA as compared with that from the livers of normal rats. Accumulated data indicate that aconitine alkaloids, in particular MA, accelerate liver RNA synthesis mainly by the increase of RNA polymerase.