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

Summary An amber fragment of the 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 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 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.     

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. IX. The role of the carboxy-terminus in enzyme assembly

Summary The assembly of RNA polymerase was studied in Escherichia coli mutants encoding large N-terminal amber fragments of the subunit. Whereas the removal of up to 20% of the carboxy-terminus does not prevent the formation of premature core enzyme, the amber fragments seem to interfere with holoenzyme production. These studies permit, therefore, the localization of a region on the polypeptide involved in sigma binding.Paper VIII is Glass et al. (1986a)     

Feedback regulation of RNA polymerase subunit synthesis after the conditional overproduction of RNA polymerase in Escherichia coli

Summary The and subunit of RNA polymerase are thought to be controlled by a translational feedback mechanism regulated by the concentration of RNA polymerase holoenzyme. To study this regulation in vivo, an inducible RNA polymerase overproduction system was developed. This system utilizes plasmids from two incompatibility groups that carry RNA polymerase subunit genes under lac promoter/operator control. When the structural genes encoding the components of core RNA polymerase (, and ) or holoenzyme (, , and ⁷⁰) are present on the plasmids, induction of the lac promoter results in a two fold increase in the concentration of functional RNA polymerase. The induction of RNA polymerase overproduction is characterized by an initial large burst of synthesis followed by a gradual decrease as the concentration of RNA polymerase increases. Overproduction of RNA polymerase in a strain carrying an electrophoretic mobility mutation in the rpoB gene results in the specific repression of synthesis off the chromosome. These results indicate that RNA polymerase feedback regulation controls synthesis in vivo.     

Variations in the rate of synthesis of beta and beta'' RNA polymerase polypeptides under the influence of certain factors.

Summary In merodiploid cells containing a double dose of structural genes of RNA polymerase subunits-rpoBand rpoC-the rate of and subunits synthesis is 2 times higher than in haploidcells. Missence mutation rpoC1 (tsX) alters polypeptide and inducesthe and 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 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 and synthesis is depressed much stronger than the total protein synthesis. Infection with a transducing phage rif ^d-47 which carries rpoB gene provokes a higher rate of synthesis. When pre-irradiated cells (500 erg/mm²) are infected with this phage, the rate of 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.     

Mutation to rifampicin resistance at the beginning of the RNA polymerase beta subunit gene in Escherichia coli

Summary The unusual recombinant plasmid pRC19 carrying the N-terminal fragment of the Escherichia coli RNA polymerase rpoB gene was found to specify high level rifampicin resistance of E. coli cells. Sequence analysis of this plasmid revealed one substitution only: transversion GT, leading to amino acid substitution Val¹⁴⁶Phe. This mutational change marks the second domain of the subunit involved in rifampicin binding.     

Primary structure of Escherichia coli RNA polymerase nucleotide substitution in the beta subunit gene of the rifampicin resistant rpoB255 mutant

Summary The transducing phage dsupM814 and the plasmid pIB1830 containing the wild-type rpoB gene have been constructed and the primary structure of the gene's central fragment has been established. In contrast with the wild-type, the gene of the rpoB255 mutant, whose primary structure has been published, was found to contain an A.T.T.A. transversion entailing the substitution of a valine residue for the aspartic acid residue (516) of the wild-type subunit.     

Biosynthesis of RNA polymerase in Escherichia coli

Summary Effect of temperature-sensitive, assembly-defective mutations in Escherichia coli RNA polymerase (rpoB) or subunit gene (rpoC) was investigated on the expression of wild-type rpoB ⁺C⁺operon, which was introduced by infection of a lambda transducing phage drif ⁺ (rpoB ⁺)-6 after UV-irradiation of the mutant cells. In rpoB2·rpoB7 strain which accumulates assembly-intermediates, free , ₂ complex and premature core, the expression of rpoB ⁺C⁺operon measured by the rate of subunit synthesis was considerably inhibited whereas that of EF(translation elongation factor)-Tu, ribosomal proteins L1 and L7/L12, and some -coded proteins remained unaffected. On the other hand, the expression was enhanced specifically for only rpoB ⁺C⁺operon in either rpoC4 or rpoC1 mutants, which are defective in the association of ₂ complex and subunit or the activation of premature core enzyme, respectively. Upon preincubation of the mutant cells at 42° C prior to phage infection, during which assembly intermediates degraded rapidly, the rate of subunit synthesis relative to other phage-corded proteins increased remarkably in rpoB2·rpoB7 mutant as well as in rpoC4 and rpoC1 mutants. These observations strongly suggested the autogenous regulation for at least (rpoB ⁺C⁺) operon by some trans-active diffusible protein complexes built of RNA polymerase subunits. Nature of the regulatory molecules is discussed.Paper VI in this series is Saitoh and Ishihama (1977)     

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

Summary In 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 + relative to total protein nor : ratio does not differ in haploid and merodiploids.     

RNA polymerase subunit biosynthesis in Bacillus subtilis

Summary The relative rates of RNA polymerase biosynthesis in Bacillus subtilis has been examined under steady-state growth conditions. The synthesis of RNA polymerase subunits (, , , ) has been followed by subunit fractionation of immunoprecipitated [³H]-labelled samples on SDS-polyacrylamide gels. The stoichiometries of ::: subunits have been determined from cultures pulse-labelled during steady-state growth. The results suggest that an unassembled pool of the -subunit exists from which the holoenzyme is formed.Upon shift-up from acetate to glycerol containing medium, a rapid rise in the differential rate of core enzyme synthesis was observed, while the rate of synthesis of the -subunit was not stimulated. During shift-down, a concomitant reduction in the rate of synthesis of all subunits occurred for the first 20 min after the shift; thereafter, a rate of synthesis characteristic of the new growth rate was established.As cultures enter sporulation, an immediate reduction in the rate of -subunit synthesis was demonstrated.     

Über den histochemischen Nachweis der β-Glueosidase mit 1-Naphthyl-β-glucopyranosid

Zusammenfassung Es wird eine einfache simultane Azokupplungsmethode zur Darstellung der -Glucosidase im Dünndarm verschiedener Säuger beschrieben und mit anderen histochemischen Verfahren zum Nachweis dieses Enzyms verglichen. Eine intrazelluläre Lokalisation der -Glucosidase ermöglichen nur die Indigogen-und die hier angegebene Technik, nicht dagegen die bisherigen Azofarbstoffmethoden mit 6-Br-2-Naphthyl--glucopyranosid als Substrat und p-Rosanilin zur Simultanoder Fast Blue B zur Postkupplung.Das Inkubationsmedium des neuen Verfahrens enthält 4,5–9,0 mg 1-Naphthyl--glucopyranosid (gelöst in 0,4 ml Dimethylformamid) und 0,4–0.8 ml 2% hexazotiertes p-Rosanilin in 9,0 ml 0,1 M Citronensäure-Phosphat-Puffer, pH 5,5. —Mikrochemische Messungen mit dem gleichen Substrat zeigen, daß die -Glucosidase durch p-Rosanilin in ähnlichem Ausmaß wie durch Ferricyanid im Indigogen-Medium gehemmt wird.Wegen der fraglichen Verwandtschaft von -Glucosidase und neutraler -Galactosidase wurde dieses Enzym mit obigem Ansatz und 1-Naphthyl--galactopyranosid als Substrat untersucht.

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On the histochemical demonstration of -glucosidase with 1-naphthyl--glucopyranoside

Summary A simple simultaneous azo coupling method for the demonstration of -glucosidase in the small intestine of various mammals is described and compared with other histochemical techniques for this enzyme. Strong evidence occurs that a correct intracellular localization of -glucosidase can only be obtained by means of the indigogenic and the assay presented here: the azo dye methods published so far with 6-Br-2-naphthyl--glucopyranoside as substrate and p-rosaniline for simultaneous or Fast Blue B for postcoupling are not able to reflect the true binding sites of intestinal--glucosidase.The recommended incubation medium consists of 4.5–9.0 mg 1-naphthyl--glucopyranoside (dissolved in 0.4 ml NN-dimethyl formamide) and 0.6–0.8 ml 2% hexazonium-p-rosaniline in 9.0 ml 0.1 M citric acid-phosphate buffer, pH 5.5.— Microchemical measurements using the same substrate show that p-rosaniline inhibits -glucosidase to a similar extent as ferricyanide in the indigogenic medium.Because of the presumed relationship between -glucosidase and neutral -galactosidase the latter enzyme has been demonstrated with the above mentioned assay replacing the 1-naphthyl--glucoside by the corresponding -galactopyranoside.The strongest -glucosidase and -galactosidase activity can regularly be observed in the jejunum of rats, mice and guinea-pigs where both enzymes are localized in the brush border region of the enterocytes. In comparison with -galactosidase the -glucosidase reaction is always more intensive and the azo dye production in the microvillous zone of suckling rats and guinea-pigs is far higher than in the intestine of adult animals. Furthermore both enzymes react in a similar way to inhibitors, experiments (thirst, hunger) and pregnancy and do not split naphthol AS BI -glucopyranoside respectively -galactopyranoside.Bloc fixation in formol-calcium and especially in glutaraldehyde improves the localization of the azo dye considerably; but microchemistry reveals that aldehyde fixation supresses the -glucosidase to ca. 50%. The basis activity of the enzyme following pretreatment with formol is reached within the first minute of fixation.

     

The energy transmission in ATP synthase: From the γ-c rotor to the α3β3 oligomer fixed by OSCP-b stator via the βDELSEED sequence

ATP synthase (F₀F₁) is driven by an electrochemical potential of H⁺ (H⁺). F₀F₁ is composed of an ion-conducting portion (F₀) and a catalytic portion (F₁). The subunit composition of F₁ is ₃₃. The active ₃₃ oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F₁ (TF₁). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the DELSEED sequence via -F₀. In fact, cross-linking of DELSEED to stopped the ATP-driven rotation of in the center of ₃₃. The torque of the rotation is estimated to be 420 pN·å from the H⁺ and H⁺-current through F₀F₁. The angular velocity () of is the rate-limiting step, because H⁺ increased theV _max of H⁺ current through F₀, but not theK _m (ATP). The rotational unit of F₀ (=ab₂c₁₀) is /5, while that in ₃₃ is 2/3. This difference is overcome by an analog-digital conversion via elasticity around DELSEED with a threshold to release ATP. The distance at the C site is about 9.6 å (2,8-diN₃-ATP), and tight Mg-ATP binding in ₃₃ was shown by ESR. The rotational relaxation of TF₁ is too rapid (=100 nsec), but the rate of AT(D)P-induced conformational change of ₃₃ measured with a synchrotron is close to . The ATP bound between the P-loop and E188 is released by the shift of DELSEED from RGL. Considering the viscosity resistance and inertia of the free rotor (-c), there may be a stator containing OSCP (= of TF₁) and F₀-d to hold free rotation of ₃₃.     

Heat shock response in Escherichia coli promotes assembly of plasmid encoded RNA polymerase beta-subunit into RNA polymerase

Summary Escherichia coli cells, carrying a rifampicin sensitive RNA polymerase -subunit gene in the chromosome and a rifampicin resistant -subunit gene placed under the control of a strong promoter in a multicopy plasmid, are unable to grow in the presence of rifampicin, despite the accumulation of large quantities of the resistant subunit. A major portion of the overproduced subunit is found in an insoluble form. Conditions known to induce the heat shock proteins (hsps), e.g. elevated temperature or the presence of ethanol in the growth medium, increase the amount of the plasmid-borne -subunit which apparently assembles into active RNA polymerase and makes the plasmid bearing cells rifampicin resistant. Alternatively, plasmid-borne subunits assemble into RNA polymerase with low efficiency in rpoH mutant cells known to have reduced level of hsps. We suggest that the plasmid-borne subunit is poorly assembled into RNA polymerase and that hsps promote the assembly by interfering with -subunit aggregation.     

The influence of mutations upon the synthesis of RNA polymerase subunits in Escherichia coli cells

Summary The influence of mutations in structural genes of and subunits of RNA polymerase upon the synthesis of these subunits in E. coli cells have been investigated. An amber-mutation ts22 in the subunit gene decreases the intracellular concentration of this subunit and the rate of its synthesis. At the same time the concentration and the rate of subunit synthesis is increased. These suggest the compensatory activation of the RNA polymerase operon that takes place under the conditions of shortage of one of the subunits. Reversions, as well as more effective supression of ts22 amber mutation, achieved by streptomycin addition, substitution of su2 by su1, or by specific mutations, result in a rise of and drop of subunit concentration and synthesis in ts22 mutant. TsX missense-mutation in the subunit gene alters the properties of the enzyme increasing, at the same time, the concentration and the rate of synthesis of both and subunits, particularly at a nonpermissive temperature. This points to an inversely proportional relationship between the rate of synthesis of RNA polymerase subunits and the total intracellular activity of the enzyme. Extra subunits are rapidly degraded in ts22 and tsX mutants.The whole complex of our data and those of others suggest that the regulation of the synthesis of RNA polymerase subunits is accomplished by interaction of a negative and a positive mechanisms of regulation which include not only activators and repressors but the enzyme itself as well.     

Non-coordinate synthesis of RNA polymerase beta beta'' subunits in a temperature-sensitive beta''-subunit mutant of Escherichia coli

Summary On exposure to high temperature of a temperature-sensitive RNA polymerase subunit (rpoC92) mutant of Escherichia coli, selective reduction was observed in the rate of synthesis of a group of proteins including RNA polymerase subunit. The finding that the synthesis of subunit but not subunit was specifically repressed in this mutant grown at non-permissive temperature indicates that the functionally intact RNA polymerase is required for the synthesis of subunits be coordinated. In addition, the assembly of newly synthesized RNA polymerase subunits was inefficient in this mutant at the steps where altered subunit was involved, and the unassembled enzyme subunits were rapidly and preferentially degraded. During recovery to non-restricted growth, the synthesis of both and subunits was transiently enhanced in parallel leading to recovery of the intracellular concentration of functional RNA polymerase.     

Identification of beta subunit of the rhesus monkey chorionic gonadotropin (rmCG)

Chorionic gonadotropin (CG) is a placental derived hormone that plays a crucial role in successful implantation and establishment of early pregnancy in the primates. The rhesus monkey was chosen as a model to understand the feasibility of developing human DNA immuno-contraceptive. The coding region of rhesus monkey CG -subunit (rmCG) was isolated by the TDRT-PCR method. The nucleotide sequence including the leader peptide was 499 nucleotide long and encoded 166 amino acids. In comparing with the previous known primates CG -subunits, the rmCG was the highest degree of homology with baboon CG -subunit at the deduced amino acid sequence (94%), 79.5% homology with human CG -subunit and 70.4% homology with marmoset monkey CG -subunit. The eukaryotic expression vector pCMV4-rmCG inserted full-coding cDNA sequence of rmCG was constructed, and the expression of rmCG -subunit in HeLa cells transient expressing system in vitro and BALB/c mice in vivo was determined. The results demonstrated that the recombinant PCMV4-rmCG eukaryotic expression vector could express rmCG -subunit in vitro and in vivo.     

Selection and analysis of non-interactive mutants in the Escherichia coli tryptophan synthase alpha subunit.

Summary The inherent infidelity of Taq DNA polymerase in the polymerase chain reaction was exploited to produce random mutations in thetrp A gene. Screening of the resulting clones allowed selection of non-interactive mutant subunits retaining their intrinsic catalytic activity. Two single changes responsible for this phenotype were identified by DNA sequencing as: 126 valine (GTG)glutamic acid (GAG) and 128 valine (GTT)aspartic acid (GAT). Three single changes giving a non-interactive phenotype with an impaired intrinsic catalytic activity were identified by DNA sequencing as a66 asparagine (AAC)aspartic acid (GAC); 109lysine (AAA) arginine (AGA); 118 cysteine (TGC)arginine (CGC). Where possible, we individually assessed the importance of these residues in interaction in light of structural information from X-ray crystallography and by intergeneric protein sequence comparison.     

Free radicals,cytokines and nitric oxide in cardiac failure and myocardial infarction

Myocardial infarction is the most common cause of congestive cardiac failure. Free radicals, cytokines, nitric oxide (NO) and antioxidants play a major role both in atherosclerosis and myocardial damage and preservation. In the early stages of atherosclerosis, neutrophils and monocytes infiltrate the intima and generate free radicals which damage the endothelial cells. As a result, production of NO and prostacyclin by the endothelial cells declines, which have cardioprotective actions. This also has relevance to the beneficial action of aspirin since, it can modulate both prostanoid and l-arginine-NO systems and NF-kB translocation. In both acute myocardial infarction and chronic congestive cardiac failure, the plasma levels of various inflammatory mediators such as interleukins and tumour necrosis factor- (TNF) are elevated. TNF, produced by the inflammatory cells and the myocardium, can suppress myocardial contractility and induce the production of free radicals, which in turn can further damage the myocardium. Transforming growth factor (TGF), polyunsaturated fatty acids and the glucose-insulin-potassium regimen can antagonize the harmful actions of TNF and protect the myocardium. This explains why efforts made to reduce the levels of pro-inflammatory cytokines have beneficial action and preserve the myocardium.     

New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids

Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: 3)--L-QuipNAc-(13)--D-GlcpNAc-(16)--D-GlcpNAc-(1 (S)-Lac-(2–3) (1) 4)--D-GlcpA-(13)--D-GalpNAc-(14)--D-Glcp-(13)--D-Galp-(14)--D-GalpNAc-(1 L-Ala-(2–6) (2) 3)--D-GalpNAc-(16)--D-GalpNAc-(14)--D-GlcpA-(1 L-Lys-(2–6)--D-GalpA-(14) (3) 4)--D-GlcpA-(16)--D-GalpNAc-(16)--D-GlcpNAc-(13)--D-GlcpNAc-(1 (R)-aLys-(2–6) (4) where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and N-[(R)-1-carboxyethyl]-L-lysine (alaninolysine), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.     

A study of the HLA-D region in patients with classic Kaposi's sarcoma

The HLA-D region in nine Sardinian patients with classic Kaposi's sarcoma was studied with two restriction enzymes, Eco RI and Eco RV, and two cDNA probes, DR and DQ. A total of 41 polymorphic restriction fragments were identified. One, an 11.5 kb Eco RV DQ fragment, was present in three of the patients but in none of the controls; a second, an 8.0 kb Eco RV DR fragment, was present in six patients and all the controls. No single fragment was identified which was significantly over or under-represented in either group.     

The histochemical localisation of hydroxysteroid dehydrogenase activity in the livers of various species

Summary In these experiments, a considerable range of hydroxysteroid dehydrogenases were demonstrated in vertebrate hepatic tissue; 3, 3, 6, 11, 16, 16, 17 and 20 were consistently present.3 hydroxysteroid dehydrogenase was fairly active in mammalian liver, but consistently greater activity was seen with the 3 dehydrogenases which are probably concerned with steroid detoxication and excretion. 6 and 11 hydroxysteroids were only moderately well used, and both these were noticeably better used in male tissue, as were also 3, 3, 16 and 16 hydroxysteroids. All mammalian liver utilised 16, 16 and 17 compounds fairly well, and 20 was consistently but poorly used.This histochemical evidence agrees with biochemical and clinical evidence for the significance and nature of steroid metabolism in the liver. Many of the enzymes showing activity in the liver have known function in the detoxication and elimination of steroids; and 3-hydroxysteroid dehydrogenase is concerned in cholesterol biosynthesis as well as the biosynthesis of progesterane. To have shown contrasting patterns of activity between liver and steroid producing endocrine tissues is further evidence for the specificity of these techniques in the study of dehydrogenase distribution.