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PubMed1976

RNA polymerase in vegetative cells of Bacillus subtilis. I. Purification and properties of RNA polymerase L1 and L2.

Two forms of RNA polymerase [EC 2.7.7.6], RPase L1 and RPase L2, isolated from a highly synchronized vegetative culture of Bacillus subtilis Marburg strain are described. RPase L1 is the major component (identical and the vegetative RNA polymerase already reported) and RPase L2 is a minor, new component corresponding to 3--5% of the total activity. The enzymes differed in their requirements for divalent ions, though the differences depended on the template DNA employed. PRase L1 is able to transcribe phage M2 DNA in the presence of Mg2+ ions and both B. subtilis DNA and phage M2 DNA in the presence of Mn2+ ions. On the other hand, RPase L2 activity can be detected only in the presence of 3 mM Mn2+ ions with all the templates. It is of interest that the transcription of phage M2 DNA by both enzymes stringently requires KC1. It may be due to this ion dependence that RPase L2 has not been detected previously. RPase L2 consists of 1beta', 1beta gamma, 1sigma, and 2alpha subunits. The molecular weight of the beta gamma subunit (about 110,000) is close to the value reported for the beta subunit of RNA polymerase prepared from sporulating cells. However, RPase L2 as a whole molecule is different from the RNA polymerase of sporulating cells or spores in the following two respects: RPase L2 contains sigma subunit as a component essential for selective transcription, and it is resistant to 1 mug of rifampicin per ml. Elimination of the sigma subunit from RPase L2 greatly stimulates RNA synthesis by the enzyme. Conversely, the addition of sigma subunit to the core-enzyme is inhibitory.

Bacillus subtilis↗
PubMed1977

Identification of an amber fragment of the beta subunit of Escherichia coli RNA polymerase: a yardstick for measuring controls on RNA polymerase subunit synthesis.

An amber fragment of the beta subunit of Escherichia coli RNA polymerase has been recovered from strains carrying the rpoB12 amber mutation, indicating that the B12 mutation resides in the structural gene for the beta subunit. The fragment is readily assayed and can be used to determine the degree of expression of a single rpoB cistron in strains haploid or diploid for this region. These studies confirm that the bacterial mechanism, which can compensate for reduced translation of the beta message, operates by the co-ordinate induction of rpoB and rpoC. Furthermore, I show that rpo control depends upon cistron(s) located on the F' factor, KLF10, whose product(s) can act negatively in trans on rpoBC expression.

DNA-Directed RNA Polymerases↗
PubMed1979

Interaction between RNA polymerase and a ribosomal RNA promoter of E. coli.

The interaction between RNA polymerase and the E. coli ribosomal (r) RNA promoter(s) of the rrnE operon has been studied by the filter-binding method. The extent of complex formation between RNA polymerase and rrnE promoter(s) is salt-dependent; ppGpp specifically inhibits interaction of RNA polymerase with the rrnE promoter(s). A tentative model is proposed for the molecular events in the early steps of rRNA initiation: a transition of the primarily formed, labile RNA polymerase-rRNA promoter complex to a more stable form is the determining step. This step is salt-sensitive; ppGpp acts on this "isomerization".

DNA-Directed RNA Polymerases↗
PubMed1976

Functional hybrid enzymes reconstituted from Escherichia coli and Serratia marcescens RNA polymerase subunits.

RNA polymerase was isolated from Escherichia coli and Serratia marcescens. The subunits of both enzymes were separated by electrophoresis on cellulose acetate sheets in the presence of urea. Under conditions favouring reconstitution of the RNA polymerases, stoichiometric amounts of the subunits were allowed to interact. Active hybrid enzymes were formed if corresponding subunits of both enzymes were mutually exchanged. The analysis of the RNA products synthesized showed that the reconstituted enzymes are able to recognize the promoters for transcription and the termination signals on the DNA template. The transcription products can serve as messengers for cell-free protein synthesis.

Binding Sites↗
PubMed1979

A relationship between DNA helix stability and recognition sites for RNA polymerase.

The RNA polymerase binding sites on the DNA of (i) the aroE-trkA-spc segment of the Escherichia coli genome, (ii) transposon Tn3, (iii) plasmid ColE1, and (iv) coliphage lambda were mapped by electron microscopy, with the use of the BAC technique; these maps were compared with the maps of the early-melting regions for the same genomes. The results indicate that in all these cases the binding sites for the E. coli RNA polymerase lie preferentially in the early melting regions of DNA. These data indicate that helix stability may be an important feature of the multipartite nature of the promoter structure.

DNA, Bacterial↗
PubMedDate not supplied

[Role of RNA-polymerase in gene activity regulation of E. coli RNA-polymerase mutants with a pleiotropic effect. I. Physiological and biochemical studies].

Four Rifr-mutants of E. coli B/r (rpo B401, rpo B402, rpo B403, rpo B409) which differ from the wild strain in one or more phenotypic properties besides rifampicin resistance were obtained. Transfer of the mutant Rifr-alleles into the parent strain gives the latter all the properties of the mutant. This indicates that the new properties are due to the pleiotropic effect of Rifr-mutations. Biochemical studies of the properties of RNA-polymerases from the mutants and the parent showed that some new properties of the mutants could not be explained by the appearance of analogous properties in the mutant RNA-polymerase itself. They seem to be caused by alteration in functional activity of the mutant enzyme, particulary, alteration of its control properties during transcription. The function of the beta-subunit in genetic transcription is discussed.

DNA, Bacterial↗
PubMed1979

Altered promoter selection by a novel form of Bacillus subtilis RNA polymerase.

Bacillus subtilis RNA polymerase holoenzyme prepared by several standard methods utilizes bacteriophage T7 DeltaD111 DNA as an efficient template. The major RNA products are specific transcripts from T7 promoters A(1) and C; these promoters are also efficiently utilized by RNA polymerases purified from a wide range of other bacterial species [Wiggs, J., Bush, J. & Chamberlin, M. (1979) Cell 16, 97-109]. In contrast, B. subtilis RNA polymerase preparations purified by a modification of the method of Burgess and Jendrisak (designated fraction 5) utilize T7 DeltaD111 promoters A(1) and C and an additional promoter site, J, which has been located at 90.6% on the standard T7 physical map. This promoter is not used by B. subtilis core RNA polymerase or by RNA polymerase from any other bacterial species we have tested. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of fraction 5 RNA polymerase shows that it contains B. subtilis components sigma and delta and a polypeptide of M(r) 92,000 in addition to the B. subtilis beta, beta', and alpha subunits. Chromatography of fraction 5 on single-stranded DNA-cellulose gives an enzyme fraction, Bs I, that is indistinguishable from B. subtilis RNA polymerase holoenzyme both in its peptide composition (betabeta'alpha(2)sigma) and in the selective transcription of only T7 RNAs A(1) and C. Chromatography of fraction 5 on phosphocellulose yields an enzyme fraction, Bs II, devoid of sigma subunit but containing the M(r) 92,000 peptide and traces of delta. This fraction synthesizes predominantly T7 J RNA in vitro together with traces of T7 A(1) and C RNAs. Hence, B. subtilis RNA polymerase fraction Bs II appears to contain a form of RNA polymerase that can transcribe selectively without detectable amounts of B. subtilis sigma subunit and that utilizes a promoter site not used by other known bacterial RNA polymerases. The structural basis for this specificity is not yet known.

Bacillus subtilis↗
PubMed1978

Glucocorticoid regulation of rat thymus RNA polymerase activity: the role of RNA and protein synthesis.

Treatment of rat thymus cells with the glucocorticoids cortisol and dexamethasone resulted in the stimulation of RNA polymerase B activity within 10 min of steroid addition. This early effect was followed by the inhibition of both RNA polymerase A and B activities. These effects were glucocorticoid-specific and were inhibited by the antiglucocorticoid cortexolone. The inhibitory effect of dexamethasone on RNA polymerase A activity was abolished by prior treatment of the cells with alpha-amanitin, cordycepin or cycloheximide, but cycloheximide was only capable of inhibiting the steroid effect measured at 3 h if added within 10--20 min after steroid addition. Cycloheximide had no effect on the steroid-mediated inhibition of RNA polymerase B activity. Control RNA polymerase A activities were unaffected by the presence of inhibitors of RNA and protein synthesis. It is concluded that the inhibition of ribosomal RNA synthesis by glucocorticoids is dependent on protein synthesis, but that basal RNA polymerase A activity in rat thymus cells is not stringently coupled to protein synthesis.

Amanitins↗
PubMed1977

Physiochemical studies on interactions between DNA and RNA polymerase. Unwinding of the DNA helix by Escherichia coli RNA polymerase.

In a medium containing 10mM Tris, pH 8, 10 mM MG++, 50 mM K+ and 10 mM NH4, the binding of an E. coli RNA polymerase holoenzyme unwinds the DNA helix by about 240 degrees at 37 degrees C. In this medium the total unwinding of the DNA increases linearly with the molar ratio of polymerase to DNA. The number of binding sites at which unwinding can occur is very large. If the K+ concentration is increased at 200 mM, the enzyme binds to only a limited number of sites, and the bound and free enzyme molecules do not exchange at an appreciable rate. The unwinding angle of the DNA per bound enzyme in this high salt medium is measured to be 140 degrees at 37 degrees C. The total unwinding angle for a fixed number of bound polymerase molecules per DNA is strongly temperature dependent, and decreases with decreasing temperature.

Coliphages↗
PubMed1976

Physicochomecial studies on interactions between DNA and RNA polymerase. Isolation and mapping of a T7 DNA fragment containing the early promoters for Escherichia coli RNA polymerase.

The cleavage sites in the early promoter region of coliphage T7 have been mapped for four restriction enzymes. They are, from the left end in base pairs, 1100 and 740 for Hinf; 680, 320, 530, 240, 77, and 67 for Hind II; 620 and 530 for Hpa II; 790 for Alu I. The nucleotide sequence between the Hind II site at 680 base pairs from the left end and the Hinf site at 740 base pairs from the left end has been determined, from which the start point of the promoter A3 is located at 720 base pairs from the left end. The start points of the other two major promoters A1 and A2 are deduced to be at 460 and 580 base pairs from the left end, respectively, from the chain lengths of the in vitro transcripts off the 1100 base-pairs long Hinf fragment. Similar to the sequences of a pL and pR promotors of phage lambda and a sequence in Simian Virus 40 used by Escherichia coli RNA polymerase as a promotor, the sequence of the A3 promotor of T7 also has a Hind II restriction site approximately 30 base pairs upstream to the start point of RNA synthesis. No such Hind II sites exist, however, for the A1 and A2 promoters. Experiments on the protection of some of the restriction sites on the 1100 base-pairs-long Hinf fragment by RNA polymerase binding support the electron microscopic observations of others that, in addition to the three sites A1, A2 and A3, there is at least a fourth site at which E. coli RNA polymerase can bind strongly. In addition to the Hind II site at 680 base pairs from the left end and the Hinf site at 740 base pairs from the left end, which are presumably protected by the binding of a single RNA polymerase at the A3 site, the Hind II site at 240 base pairs from the left end is also protected at a level of 5 polymerase molecules/DNA fragment. The possible existence of several minor promotor sites in the early promotor region, in addition to the three major promotor sites, is discussed.

Base Sequence↗
PubMed1977

A gene from Escherichia coli affecting the sigma subunit of RNA polymerase.

The RNA polymerase sigma subunits of Escherichia coli K, E. coli C, and Salmonella typhimurium can be resolved by electrophoresis. Using this technique, we have analyzed Salmonella strains carrying F' plasmids from E. coli K in order to map the gene for the sigma factor. Partial diploid analyses show the location of the sigma gene at 62-66 min on the E. coli genetic map. This gene is cotransducible with toIC and dnaG, at 66 min.

Chromosome Mapping↗
PubMedDate not supplied

[Effect of oligoribonucleotides specifically bound by E. coli RNA-polymerase on DNA-dependent RNA synthesis].

It was shown previously that E. coli RNA-polymerase (EC 2.7.7.6) selectively binds certain fractions of penta- or hexaribonucleotides random mixtures (Knorre V. L., Vasilenko S. V., Salganik R. I., FEBS Lett., 30, 229, 1973). The data obtained demonstrate that such oligoribonucleotides compete with DNA for the RNA polymerase active centre and inhibit DNA dependent RNA synthesis catalyzed by the enzyme. These properties are absent in tri- and tetraribonucleotides which cannot be bound by RNA polymerase. The inhibitory action of the pentaribonucleotides was higher when they had been added prior to DNA to the mixture containing RNA polymerase.

DNA-Directed RNA Polymerases↗
PubMed1976

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

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.

Amanitins↗
PubMed1975

Synthesis of specific functional messenger RNA in vitro by phage-SP01-modified RNA polymerase of Bacillus subtilis.

RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was purified from rifampicin-resistant Bacillus subtilis, from both uninfected cells and cells infected with bacteriophage SP01. The enzyme from infected cells lacked all traces of the sigma subunit, contained several polypeptides absent from the enzyme made in uninfected cells, and had an altered template specificity in a transcription assay. A cell-free protein synthesizing system from Escherichia coli, when poisoned with rifampicin, was completely dependent on addition of either of these RNA polymerase preparations for DNA-dependent protein synthesis. Under these conditions, the SP01-modified RNA polymerase preferentially stimulated the synthesis of functional mRNA for the phage enzyme dCMP deaminase (deoxycytidylate aminohydrolase, EC 3.5.4.12), whereas unmodified B. subtilis RNA polymerase could stimulate synthesis of this mRNA in small quantity and only after prolonged incubation. This mRNA belongs to a class of phage transcripts (m) which cannot be transcribed in vivo in the absence of phage-specific protein synthesis.

Aminohydrolases↗