Showing papers on "RNA polymerase III published in 1976"

RNA polymerase specificity and the control of growth.

Abstract: RNA polymerase is an allosteric enzyme whose activity and specificity are controlled by interaction with a nucleotide, tRNA and proteins In E coli the availability of these effectors should ensure that the quality of transcription is tightly coupled both to translation and to the metabolic state of the cell

Molecular structure of yeast RNA polymerase III: demonstration of the tripartite transcriptive system in lower eukaryotes.

Abstract: Homogeneous RNA polymerase III (RNA nucleotidyltransferase III) has been obtained from yeast. The subunit composition of the enzyme was examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is composed of 12 putative subunits with molecular weights 160,000, 128,000, 82,000, 41,000, 40,500, 37,000, 34,000, 28,000, 24,000, 20,000, 14,500, and 11,000. The high-molecular-weight subunits and several of the smaller subunits of yeast RNA polymerase III are clearly different from those of enzymes I and II, indicating a distinct molecular structure. However, the molecular weights of some of the small subunits (41,000, 28,000, 24,000, and 14,500) appear to be identical to those of polymerases I and II. Thus, it is possible that the three classes of enzymes in yeast have some common subunits. As in other eukaryotes, yeast polymerase II is inhibited by relatively low concentrations of alpha-amanitin; however, contrary to what has been found in higher eukaryotes, yeast polymerase III is resistant (up to 2 mg/ml) to alpha-amanitin, while yeast polymerase I is sensitive to high concentrations of the drug (50% inhibition at 0.3 mg/ml). These results establish the existence of RNA polymerase III in yeast and provide a structural basis for the discrimination of the three functional polymerases in eukaryotes.

Transcription in yeast: alpha-amanitin sensitivity and other properties which distinguish between RNA polymerases I and III

Abstract: Three peaks of DNA-dependent RNA polymerase (RNA nucleotidyltransferase) activity are resolved by chromatography of a sonicated yeast cell extract on DEAE-Sephadex. The enzymes, which are named RNA polymerases I, II, and III in order of elution, show similar catalytic properties to the vertebrate class I, class II, and class III RNA polymerases, respectively. Yeast RNA polymerase III is readily distinguished from yeast polymerase I by its biphasic amnonium sulfate activation profile with native DNA templates, greater enzymatic activity with poly[d(I-C)] than with native salmon sperm DNA, and distinctive chromatographic elution positions from DEAE-cellulose (0.12 M ammonium sulfate) compared with DEAE-Sephadex (0.32 M ammonium sulfate). The three yeast RNA polymerases also show significant differences in alpha-amanitin inhibition. RNA polymerase II is the most sensitive (50% inhibition at 1.0 mug of alpha-amanitin per ml). Contrary to the results for vertebrate systems, yeast polymerase I can be completely inhibited by alpha-amanitin at high concentrations (50% inhibition at 600 mug/ml) while yeast RNA polymerase II BEGINS TO SHOW SIGNIFICANT INHIBITION ONLY AT CONCENTRATIONS EXCEEDING 1 MG/ML. Therefore, yeast RNA polymerases I and III show a pattern of alpha-amanitin sensitivity that is the reverse of that seen for the analogous vertebrate RNA polymerases.

Transcription of specific genes in isolated nuclei from HeLa cells in vitro

Abstract: Isolated HeLa cell nuclei were employed to catalyze the synthesis of RNA in vitro. In the presence of low concentrations of alpha-amanitin (1 mug/ml), used to suppress the formation heterogeneous nRNA, these nuclei synthesize RNA very efficiently for extended periods of time (at least 60 min) at an elongation rate of about seven nucleotides per second. The product, analyzed on sucrose density gradients and polyacrylamide gels was found to exist of two predominant size classes. Synthesis of the 45-S ribosomal precursor was completely resistant even to high concentrations of alpha-amanitin (150 mug/ml) and hence was catalyzed by enzyme A (or I). A limited degree of processing of the 45-S precursor occurred in vitro. In addition, a second RNA class of low molecular weight (4-8 S) was synthesized by HeLa cell nuclei in the presence of 1 mug/ml alpha-amanitin in vitro. Analysis on 8% polyacrylamide gels resolved the RNA into four distinct components. Their synthesis was resistant to low (1 mug/ml) but clearly sensitive to high (150 mug/ml) concentrations of alpha-amanitin. Consequently the synthesis of all these small-molecular-weight RNA species is catalyzed by RNA polymerase C (or III). For the assessment of the initiation frequency of the individual classes of RNA, a new technique was developed independent of labelling the 5' end of the RNA molecule with the gamma-phosphate of the initiating nucleotide. It employs the double labelling of an RNA molecule with two different isotopes added sequentially at different stages of completion of the chain. From the incorporation ratio of the two isotopes into a particular class of RNA, conclusions can be drawn concerning their initiation frequency. The results obtained have shown a high reinitiation frequency for the small-molecular-weight RNA species at all stages of the incubation reaction. In contrast, reinitiation of the 45-S precursor RNA occurs only to a limited extent in isolated HeLa cell nuclei in vitro.

REPLICAtion of small plasmids in extracts of Escherichia coli: requirement for both DNA polymerases I and II.

Abstract: The role of the three E. coli DNA polymerases (pol I, II, and III) in the replication of Col E1 DNA and other small plasmids with similar replicative properties was investigated in a soluble in vitro system prepared by freeze-thaw lysis of chloramphenicol-treated cells (Staudenbauer, 1976). Extracts from isogenic mutants of the polA, polB and polC gene loci deficient in pol I, II, and III respectively were examined for their replicative capacity. It was found that polA and polC extracts are deficient in the synthesis of supercoiled plasmid DNA, whereas the polB mutation has not effect. Deficient extracts could be complemented by addition of purified pol I and pol III holoenzyme. Analysis of the in vitro synthesized DNA by alkaline gradient centrifugation indicates that pol I is involved in an early step of the replication cycle whereas pol III is required at a later stage. These conclusions are confirmed by inhibition studies employing arabinosylcytosine triphosphate (aCTP) which is shown to interfere with pol III as well as pol II. The strong inhibitory effect of aCTP on plasmid replication is not influenced by the polB mutation and mimicks the effects of thermal inactivation of polC extracts. It is suggested that aCTP blocks plasmid DNA replication in vitro by interfering with pol III function.

Specific binding of formylated initiator-tRNA to Escherichia coli RNA polymerase

Abstract: E. coli fMet-tRNAfMEet and E. coli RNA plymerase (RNA nucleotidyltransferase; EC 2.7.7.6; nucleoside-triphosphate:RNA nucleotidyltransferase) form a 1:1 complex with an apparent association constant of 9.0 X 10(6)M-1 at 37 degrees. The affinity of polymerase to tRNA depends on the tRNA as well as the formyl methionine moiety. Core polymerase has a greatly reduced affinity for initiator tRNA. Optimal binding conditions are similar to those that are also optimal for binding initiator tRNA to ribosomes. Binding of initiator tRNA to polymerase stimulates the transcription of lambda plac DNA, as determined in a crude cell-free system for beta-galactosidase (EC 3.2.1.23; beta-D-galactoside galactohydrolase) synthesis as well as in a highly purified transcription system.

Purification and Characterization of DNA-dependent RNA Polymerases from Cauliflower Nuclei

Abstract: DNA-dependent RNA polymerases were solubilized from nuclei of cauliflower inflorescences and purified by agarose A-1.5m, DEAE-cellulose, DEAE-Sephadex, and phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerases I + III were separated from II by DEAE-cellulose chromatography. Subsequent chromatography on DEAE-Sephadex resolved RNA polymerase I from III. RNA polymerases I and II were further purified to high specific activity by phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerase I was refractory to α-amanitin at 2 mg/ml. RNA polymerase II was 50% inhibited at 0.05 μg/ml, and RNA polymerase III was 50% inhibited at 1 to 2 mg/ml of α-amanitin. The enzymes were characterized with respect to divalent cation optima, ionic strength optima, and abilities to transcribe cauliflower, synthetic, and cauliflower mosaic virus DNA templates.

Nucleic acid polymerizing enzymes in developing Strongylocentrotus franciscanus embryos.

Abstract: DNA-dependent RNA polymerase, DNA-Dependent DNA polymerase, and terminal riboadenylate transferase (TRT) activities have been measured after DEAE-Sephadex chromatography of whole cell extracts prepared from eggs and staged embryos of the urchin, Stronglyocentrotus franciscanus. Activity of each of these three polymerase classes is present in the egg, and the total activity per embryo is constant throughout embryogenesis to the pluteus stage (approximately 1000 cells). Thus the egg appears to contain sufficient DNA polymerase, RNA polymerase, and TRT TRT for embryogenesis. The increases in the synthesis of DNA, RNA and polyadenylated RNA tracts observed after fertilization must be due to the activation of the preexisting egg enzymes. Separation of the egg into nucleate and anucleate halves demonstrates that the RNA polymerases are not restricted to the egg nucleus. During development, the enzymes become progressively more associated with the cell nucleus. The egg extracts contain low activities (approximately 6% total) of RNA polymerase II as measured by sensitivity to alpha-amanitin. This is confirmed by resolution of the RNA polymerase forms I, II, and III by gradient sievorptive elution on DEAE-Sephadex. Later stage embryos contain more nearly equal activities of RNA polymerase, I, II, and III, although the total RNA polymerase activity per embryo is not changed. Additionally, two chromatographicallly distinct species of RNA polymerase III are detected, one of which is observed only in later stages. Thus interconversion of enzymes via addition of new subunits or coordinate synthesis and loss of enzyme species must occur.

A bacterial RNA polymerase mutant that renders λ growth independent of the N and cro functions at 42°C

Abstract: We describe a bacterial RNA polymerase mutation, rif 501, which confers rifampicin resistance and thermosensitivity to E. coli K12. The purified RNA polymerase enzyme from rif 501 bacteria shows increased heatsensitivity in vitro at 51°C. However, in vivo, at 42°C, the non-permissive temperature, mutant bacteria continue to grow and to synthesize RNA for 90 min.

Extended RNA synthesis in isolated nuclei from rat pituitary tumor cells.

Abstract: Nuclei of GH3 cells, isolated by detergent lysis, synthesized RNA for an extended period at 29 degrees C in the presence of rat liver ribonuclease inhibitor (RI). Extended RNA synthesis was dependent upon the presence of RI. Sucrose gradient sedimentation analysis of the cell-free reaction products showed that RNAs ranging from 4 S to greater than 28 S were synthesized. Further characterization of the RNA products was made by examining the sensitivity of synthesis to alpha-amanitin and actinomycin D as well as by oligo(dT)-cellulose binding properties. Evidence was obtained that RNA polymerases I, II, and III were functioning in isolated GH3 nuclei. Newly synthesized RNAs were found in both the nuclear pellet and postnuclear supernatant fractions. RNA polymerase I products remained associated with the nuclear pellet throughout a 60-min incubation period whereas RNAs synthesized by RNA polymerase III emerged rapidly into the supernatant fraction. RNA polymerase II products were distributed in both fractions and were found to contain poly(A). De novo poly(A) synthesis was demonstrated and found to be inhibited by cordvcepin triphosphate (3'-dATP). Supernatant RNAs synthesized by polymerase II contained a poly(A) segment of about 150 adenine residues; these transcripts sedimented heterogeneously with an apparent size distribution (under denaturing conditions) which was smaller than that of nuclear RNA polymerase II products and which resembled that of cellular mRNA. Qualitative differences in the nuclear and supernatant RNAs, the kinetics of appearance of the latter, and the differential effect of 3'-dATP on the extranuclear appearance of supernatant RNAs suggest that a process resembling nuclear-cytoplasmic RNA transport occurred in this cell-free nuclear system.

Bacillus Subtilis RNA Polymerase and its Modification in Sporulating and Phage‐Infected Bacteria

Abstract: Bacillus subtilis RNA polymerase holoenzyme consists of the subunits beta', beta, sigma, alpha, delta, and omega. In sporulating bacteria and in bacteria infected with phages SP01 and SP82, this enzyme undergoes changes in subunit composition and transcriptional specificity that could play a regulatory role in gene transcription. Sporulating bacteria may contain a specific component that inhibits the activity of the sigma subunit of polymerase probably by interfering with the binding of sigma-polypeptide to core enzyme. The hypothetical inhibitor may be metabolically unstable, since its activity is rapidly depleted from sporulating cells in the presence of chloramphenicol. Inhibition of sigma-polypeptide activity may restrict the transcription of phage DNA an infected sporulating cells. Although lacking the sigma-subunit, RNA polymerase purified from sporulating cells contains sporulation-specific subunits of 85,000 and 27,000 daltons. In SP01-infected bacteria, the sigma-subunit is replaced by phage-induced subunits. Purified enzyme containing the protein product of SP01 regulatory gene 28 directs the transcription of phage middle genes in vitro, while enzyme containing phage-induced polypeptides V and VI preferentially copies late genes. Accurate transcription of middle and late genes in vitro requires the host delta-subunit of polymerase (or high ionic strength) but not sigma-subunit. Phage PBS2 induces an entirely new multisubunit RNA polymerase that specifically transcribes PBS2 DNA in vitro. This enzyme is synthesized de novo after infection and does not arise by modification of the B. subtilis holoenzyme.

Phage T4 infection restricts rRNA synthesis by E. coli RNA polymerase.

Abstract: RNA polymerase from T4 infected cells supplemented with E. coli σ polypeptide has a lower affinity for rRNA promoters than RNA polymerase from uninfected cells. The pattern of transcription by the phage modified polymerase is qualitatively similar to that of the vegetative polymerase in the presence of ppGpp. We suggest that E. coli polymerase holoenzyme normally exists in at least two conformational states, one with a high affinity for rRNA promoters and another with a low affinity, and that T4 infection stabilises the low affinity form.

The Tyrosine tRNA Promoter

Abstract: INTRODUCTION The DNA of the transducing bacteriophage φ 80psu III + has been used in recent years in studies of the transcription of the tRNA 1 Tyr gene by the RNA polymerase in vitro (Daniel et al. 1970; Ikeda 1971; Zubay, Cheong and Gefter 1971). The transcript containing the tRNA sequence was invariably found to be much larger (up to 1000 nucleotides) than the expected tRNA molecule. On the other hand, Altman and Smith (1971) isolated a tRNA 1 Tyr precursor of 129 nucleotides from E. coli cells infected with φ 80psu III + . This precursor contained a 5′ pppG end group, which immediately showed that the product contained an in vivo initiation point, and second, that the initiation occurred 41 nucleotides beyond the 5′ end of the native tRNA (Altman and Smith 1971). Whether the purified RNA polymerase recognizes the same promoter in vitro and is able to initiate transcription with the same sequence as in vivo could not be clearly demonstrated in the previous work because of the background transcription of the bacteriophage genes and the length of the in vitro tRNA precursor. A defined study free from the above complications would be possible using DNA fragments that contain only the tRNA gene with its promoter as a template for RNA polymerase. It has been shown (Landy, Foeller and Ross 1974) that the DNA of the bacteriophage φ 80psu III + can be cut by the restriction enzymes Hind II + III in such a way that the tRNA 1 Tyr gene remains intact. Furthermore, upstream of the 3′ end of the tRNA...

Levels of RNA polymerase activities during growth, encystment and germination ofPhysarum polycephalum.

Abstract: Two RNA polymerases, enzyme A and enzyme B, were prepared fromPhysarum plasmodia. They are not located in the cytosol. Isolated nuclei, however, contain only a fraction of the total RNA polymerase activity: 1.5–19% depending on the nuclear preparation method.

Selective transcription of the 5s rna genes in isolated chromatin by rna polymerase iii

Abstract: Previous studies have shown that the genes which encode the ribosomal 5S RNA and the tRNAs are transcribed by a class III enzyme(s) in somatic cells. To investigate the mechanism and regulation of RNA polymerase III function, the in vitro transcription of the 5S RNA genes expressed in X. laevis oocytes is being analyzed. The RNA polymerase III from these oocytes has been purified to homogeneity and has a complex subunit structure very similar to that of somatic cell class III enzymes. The present studies suggest that the oocyte RNA polymerase III does not accurately transcribe the 5S RNA genes present in a purified recombinant plasmid DNA, even though the 5S DNA may be more efficiently transcribed than the adjacent bacterial plasmid DNA sequences. Strikingly different results were obtained when chromatin from immature X. laevis oocytes, active in 5S RNA synthesis, was used as the template for homologous RNA polymerase III. Although this chromatin contains endogenous RNA polymerase III activity, the level of total RNA synthesis and the level of 5S RNA synthesis were stimulated 10-50 fold by exogenous RNA polymerase III. Furthermore, transcription of the 5S RNA genes present in chromatin was highly selective (3000-fold above random) and predominantly asymmetric. X. laevis oocyte RNA polymerase I stimulated total RNA synthesis from this chromatin about twofold but did not stimulate transcription of the 5S genes. It is concluded that both chromosomal proteins and a purified homologous RNA polymerase III are necessary and sufficient for selective transcription of the 5S RNA genes in vitro .

Molecular Structures of Eukaryotic Class III RNA Polymerases

Abstract: INTRODUCTION DNA-dependent RNA polymerase III represents one of the three major classes of nuclear RNA polymerases in eukaryotes (Roeder and Rutter 1969). The class III enzymes have characteristic catalytic and chromatographic properties which distinguish them from the class I and II enzymes (Roeder and Rutter 1969; Adman, Schultz and Hall 1972; Roeder 1974a). In animal cells, these enzymes are also distinguished by their unique α -amanitin sensitivities (Schwartz et al. 1974; Sklar and Roeder 1975; Seifart and Benecke 1975; Weil and Blatti 1975). Although lower eukaryotes may contain only a single chromatographic form of RNA polymerase III (Roeder and Rutter 1969; Adman, Schultz and Hall 1972), multiple chromatographic forms have been described in a variety of mammalian cell types (Schwartz et al. 1974; Jaehning, Stewart and Roeder 1975; Weinmann et al. 1976; Sklar and Roeder 1975, 1976; Seifart and Benecke 1975). In animal cells, a class III RNA polymerase(s) has been implicated in the synthesis of tRNA and 5S RNA species (Weinmann and Roeder 1974) and in the synthesis of low molecular weight viral RNAs (Weinmann, Raskas and Roeder 1974; Weinmann et al. 1976; Jaehning et al., this volume). Although RNA polymerase III usually accounts for only a small proportion of the total cellular RNA polymerase activity, it has been detected, using appropriate analytical methods, in all tissues examined (Schwartz et al. 1974). However, the cellular levels of RNA polymerase III vary among different cell types and in the same cell type under different physiological conditions (Roeder 1974b; Schwartz et al....

The role of polymerase III in conjugation between E. coli K12 donor and recipient strains carrying dnaE ts mutation.

Abstract: The possible role of DNA polimerase III in conjugation was studied in a series of mutants temperature-sensitive for DNA polymerase III synthesis. The temperature-sensitive DNA mutation called dnaE 486 (ts) prohibits vegetative DNA replication at 41-45 degrees. Transfer of episome and chromosome from temperature-sensitive donor, carrying dnaE mutation to wild-type recipient strains, revertants and dnaE recipients was investigated. In the first two cases the number of Lac+ sexductants being even slightly higher at 43 degrees. Conjugational synthesis accompanying transfer involving the combination of dnaE (ts) thymine dependent and thymine independent donor and recipient strains measured by incorporation of 14C thymine was observed at the restrictive temperature. In the case of conjugation with temperaturesensitive recipient strains a drop of Lac+ sexductants and Pro+ recombinants may be as a result of disturbances in the synthesis of complementary strand in recipient, known to be dependent on pol III. However, the episome investigated by centrifugation in neutral CsC1 gradient after its transfer to the recipient with faulty polymerase III was double stranded (replicated) at the restrictive temperature.

Purification of DNA‐Dependent RNA Polymerase C from Zajdela Hepatoma Cells

Abstract: A C-type DNA-dependent RNA polymerase has been extracted and purified from the cytoplasmic fraction of Zajdela hepatoma cells. The purification method and the separation from other RNA polymerase are described. RNA-polymerizing properties of this RNA polymerase C are studied and compared with those of other RNA polymerases. We reach the conclusion that the cytoplasmic C enzyme is clearly different from A and B nuclear enzymes and that the nuclear RNA polymerase III and the cytoplasmic RNA polymerase C behave like closely related enzymes.