Zh. M. Gorlenko

Studies on the functions of the RNA polymerase components by means of mutations.

SummaryIt had been shown earlier, that RNA polymerase 13 S particles contain the large components with a molecular weight of about 3–105 and small subunits with a molecular weight of 4·104-1·105. These polymerase components easily dissociate and reassociate with restoration of the enzyme activity.Both temperature-sensitive (tsX) and rifamycin-resistant (rif-r-I) mutations proved to affect the large polymerase component without changing the small subunits. These mutations were mapped at different, though closely linked, loci of metB-thi region of E. coli K12 chromosome. These results as well as certain literature data allow to conclude that the large RNA polymerase component consists of at least two polypeptides, one being altered by ts mutation, and the other—by rif-r mutation.The large polymerase component when separated from the small subunits retain the ability to bind to T2 phage DNA while the separate small subunits lack this property. Rifamycin does not affect RNA polymerase-T2 DNA binding while ts mutation leads to inability of the enzyme to form stable complexes with DNA. Therefore, it is likely that the polypeptide affected by ts mutation is responsible for the attachment of RNA polymerase to specific sites of DNA template. On the other hand, the small subunits as well as polypeptide of the large component, which determines RNA polymerase sensitivity to rifamycin, seem not to participate in the enzyme binding to DNA template. It is suggested, that the catalytic site of RNA polymerase is located in the large component and formed by rifamycin-binding polypeptide. The small subunits are supposed to have regulatory function and activate the large components.

Interaction of RNA polymerase mutations in haploid and merodiploid cells of Escherichia coli K-12.

SummaryCombination in the same chromosome of tsX mutation which affects the attachment of RNA polymerase to DNA template with either of two rifampicin resistant mutations (rif-r-1 or rif-r-5) is lethal. However tsX forms viable combination with other rifampicin resistant mutation—rif-r-76. Moreover a partial restoration of rifampicin binding capacity takes place: RNA polymerase from double tsX rif-r-76 mutant binds rifampicin better than the enzyme from tsX+ rif-r-76 cells. No mutual influence of rifampicin resistant and streptolydigin resistant mutations was found.Heterozygous merodiploids (rif-r/rif-s and stl-r/stl-s) demonstrate phenotypic dominance of sensitivity to each of the drugs no matter whether resistant allele is localized in chromosome or in episome. However certain chromosomal mutations which themselves have no apparent effect on RNA polymerase may cause dominance of rif-r allele.About a half of total cellular RNA polymerase in crude extracts of rif-r/rif-s and stl-r/stl-s heterogenotes was found to be drug-resistant, though rif-s allele is dominant phenotypically.The development of T2 phage is completely inhibited by rifampicin in haploid rif-s cells and is only slightly affected in rif-r mutant. A partial resistance of phage development to rifampicin was observed in rif-r/rif-s heterogenotes which confirms that both rif-s and rif-r enzymes are simultaneously present in such cells.Sensitive and resistant RNA polymerase function independently when mixture of the two enzymes was incubated with the excess of DNA template. However a competition between the two enzymes for the DNA was observed if the limiting amount of the template is available. The result of this competition to major extent depends on which of the enzymes was added first. It is supposed that in certain conditions normal RNA polymerase may act as a repressor of the mutant enzyme: drug-sensitive RNA polymerase may bind to the template in the presence of the drug and thus prevent the function of drug-resistant enzyme. This hypothesis explains phenotypic dominance of sensitive alleles to resistant alleles which leads to inability of heterogenote cells to multiply in the presence of corresponding drugs.

Studies on the RNA polymerase in Escherichia coli K12 using the mutation affecting its activity.

Abstract RNA polymerase has been purified from the mutant ts-19 of Escherichia coli K12, which does not synthesize RNA at elevated temperatures. RNA polymerase from this strain was unable to synthesize complementary RNA on a DNA template in vitro . Studies of the complementation of the mutant polymerase with the normal E. coli polymerase inactivated by moderate heating were carried out. It has been found that mixing these two inactive polymerase preparations resulted in the appearance of DNA-dependent RNA synthesis, reaching 10 to 30% of the activity of the native enzyme. It was concluded that the mutation and heating affect different subunits of the enzyme and undamaged components can complement in vitro . Heated wild-type polymerase could also be activated by the addition of protein fraction eluted with 0.4 m -KCl from the DEAE cellulose column during standard polymerase purification. This protein was called “restoring protein”. Our results lead to the conclusion that RNA polymerase is built up of different components which can spontaneously re-associate giving active polymerase molecules: (1) the larger component with a molecular weight about 300,000, which is not inactivated by moderate heating but is affected by the mutation; and (2) smaller subunits which aro damaged by heating but are not inactivated by the mutation ts-19.

DNA supercoiling and transcription in Escherichia coli: influence of RNA polymerase mutations.

SummaryCoumermycin A1, a specific inhibitor of DNA gyrase, differentially changes the spectrum of proteins synthesized in wild type E. coli cells but has no effect on the protein spectrum in mutant cells with coumermycin-resistant DNA gyrase. The rpoB265 mutation affecting RNA polymerase decreases the coumermycin A1-sensitivity of bacteria while the rpoC3 mutation increases it. The interaction of wild type and mutant RpoB265 RNA polymerases with ColE1 plasmid DNA in vitro is differently affected by DNA supercoiling. No such differences are observed in the case of RpoC3 RNA polymerase. The results suggest that template supercoiling may have a substantial effect on transcription in vivo, an effect which, in some cases, depends on the properties of RNA polymerase.

Temperature sensitive mutations affecting RNA synthesis in Escherichia coli

SummaryA streptomycin method has been used for the isolation of mutants with RNA synthesis inhibited at elevated temperature. The method is based on the observation that streptomycin kills bacteria with normal RNA synthesis and does not affect the cells with inhibited synthesis of RNA. This selection method increases the yield of temperature sensitive mutants by a factor 10–20, the amount of mutants with disturbed RNA synthesis is increased 3–5 fold as compared with the method of replicas.Several types of mutants were found among the temperature sensitive strains: those possessing temperature sensitivity of one, two or three types of cellular macromolecules DNA, RNA and protein. The screening among the mutants with affected RNA synthesis revealed a strain ts-19 showing low RNA polymerase activity in cell extracts and partially purified RNA polymerase preparations. The presented evidence suggests that ts-19 mutation affects the structural gene of one of the RNA polymerase subunits.The mapping of the corresponding locus indicated that it was located between the str and thy loci in E. coli K 12 chromosome at a distance of about 20 recombination units from the first locus.

Localization of streptolydigin resistant mutation in E. coli chromosome and effect of streptolydigin on T2 phage development in stl-r and stl-s strains of E. coli.

Abstract The order of mutations affecting RNA polymerase is thi - rif - stl-argH . Transduction experiments revealed high frequency of recombination between rif and stl markers (about 50 per cent). Streptolydigin as well as rifampicin inhibits phage T2 development in the host cells sensitive to the drugs but not in rif-r stl-r host cells.

The effect of rifampicin upon the transcription of RNA polymerase β-gene in Escherichia coli

SummaryWe studied the rate of synthesis of β- and β′-subunits of DNA-dependent RNA polymerase and the rate of β-polypeptide mRNA synthesis in rifampicin-treated bacteria. The chosen antibiotic doses did not significantly inhibit the total RNA and protein synthesis in rifampicin-sensitive bacteria. For RNA-DNA hybridization experiments a pOD162 plasmid was constructed carrying a fragment of the rpoB gene and no other chromosome DNA regions. It was found that low doses of rifampicin cause an absolute and a relative increase in the rate of synthesis of the specific mRNA for the β-subunit, suggesting a stimulation of the corresponding gene transcription and excluding the possibility of a less pronounced inhibition of the rpoB gene expression compared to that of most other genes. However the relative acceleration of transeription is substantially higher than the absolute one.The stimulating effect of rifampicin on the β-polypeptide synthesis is also demonstrated in a coupled system of transcription and translation directed by λ rifd47 DNA.The possible mechanisms of the rifampicin action are discussed.

Variations in the rate of synthesis of ? and ?? RNA polymerase polypeptides under the influence of certain factors

SummaryIn merodiploid cells containing a double dose of structural genes of RNA polymerase subunits-rpoBand rpoC-the rate of β and β′ subunits synthesis is 2 times higher than in haploidcells. Missence mutation rpoC1 (tsX) alters β′ polypeptide and inducesthe β and β′ subunits synthesis at increased rate, particularly at a nonpermissive temperature. When rpoBCoperon carrying mutation rpoC1 is duplicated no dosage effect is observed. In the rpoC+/rpoC1 heterodiploid the rpoC1 mutation does not significantly accelerate RNA polymerase subunits synthesis i.e. is recessive with respect to rpoC+ Rifampicin causes 6-fold stimulation of RNA polymerase subunits synthesis in a sensitive wild-type strain. The rpoC1 mutation itself accelerates the synthesis of these subunits 3-fold. In the presence of rifampicin the mutant strain produces ββ′ 13–22-fold faster as compared to wild-type strain without the drug. Thus, the effects of rifampicin and the mutation are multiplied suggesting that these factors act independently. Similar data have also been obtained with rifampicin-treated cells of rpoB22 (ts22) amber-mutant.After UV-irradiation of cells β and β′ synthesis is depressed much stronger than the total protein synthesis. Infection with a transducing phage λ rifd-47 which carries rpoB gene provokes a higher rate of β synthesis. When pre-irradiated cells (500 erg/mm2) are infected with this phage, the rate of β synthesis grows 20-fold compared to irradiated, non-infected cells and 6.5-fold compared to intact cells.The data are discussed in terms of the possible regulatory mechanisms of RNA polymerase subunit synthesis.

Content of RNA polymerase in haploid and merodiploid strains of Escherichia coli

SummaryIn cell-free extracts of E. coli merodiploids carrying F-factor with ilv-thi chromosome fragment the activity of RNA polymerase is not increased, and there is no excess of free active core-enzyme or sigma-factor. Only immunochemical analysis reveals 25% excess of RNA polymerase material in some merodiploids as compared to a haploid. However, neither the amount of β+β′ relative to total protein nor β:β′ ratio does not differ in haploid and merodiploids.

A nonconjugative mobilizable broad host range plasmid of Acinetobacter sp. that determines HgCl2 resistance

SummaryHgCl2-resistant strains of Acinetobacter sp. obtained from the soil at the Khaidarkan mercury mine (Kirghiz SSR) were found to contain, apart from large plasmids (∼60 kb), a small plasmid (∼7.5 kb) designated pKL1. It was established by conjugative crosses and transformation that pKL1 is a broad host range mobilizable plasmid and that it carries the Hgr determinant. The restriction map of pKL1 was constructed; the site of the Hgr determinant and the regions essential to replication were localized. A comparison of these results with earlier data suggests that microorganisms belonging to one microbiocenosis may carry Hgr determinants on plasmids with highly different structures and properties.