Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of sigma 70 and sigma 38.

The intracellular levels of two principal sigma subunits, sigma 70 (sigma D, the rpoD gene product) and sigma 38 (sigma s, the rpoS gene product), in Escherichia coli MC4100 were determined by a quantitative Western immunoblot analysis. Results indicate that the level of sigma 70 is maintained at 50 to 80 fmol per micrograms of total proteins throughout the transition from the exponential growth phase to the stationary phase, while the level of sigma 38 protein is below the detection level at the exponential growth phase but increases to 30% of the level of sigma 70 when cell growth stops to enter into the stationary phase. Beside the stationary phase, the increase in sigma 38 level was observed in two cases: exposure to heat shock at the exponential phase and osmotic shock at the stationary phase.     

Macromolecule synthesis in Escherichia coli BB under various growth conditions.

The kinetic behavior of the macromolecule synthesis of Escherichia coli during balanced growth in various media at different temperatures as investigated. The results indicate that macromolecule contents per cell can be expressed as exponential functions of the specific growth rate at a given temperature. It was shown that the content per cell at the zero growth rate was constant in each macromolecule component, irrespective of the growth temperature. The rate of ribonucleic acid (RNA) synthesis per unit weight of deoxyribonucleic acid and that of protein synthesis per unit weight of RNA were taken as efficiencies of RNA and protein synthesis, respectively; both of them were found to be dependent on the growth rate and temperature. The efficiency of RNA synthesis was found to be very high at a high growth rate, whereas that of protein synthesis was found to decrease above certain growth rate. At the same growth rate, an increase in the growth temperature resulted in a decrease in the efficiency of RNA synthesis but an increase in that of protein synthesis.     

Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase

We have constructed a plasmid that overexpresses 100-fold the sigma subunit of Escherichia coli RNA polymerase. The plasmid was constructed by placing the pLoL promoter-operator of bacteriophage lambda upstream from rpoD, the gene encoding the sigma subunit. A simple procedure for purification of the overexpressed protein has been developed based on guanidine hydrochloride denaturation/renaturation, DEAE cellulose chromatography, and Sephacryl S-200 chromatography. The purified product has been characterized and found to be indistinguishable from normally expressed sigma protein purified by previous protocols as judged by enzymatic activity, heat inactivation, and partial proteolysis.     

Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase.

An improved purification procedure is described for the sigma subunit of escherichia coli DNA-dependent RNA polymerase [ribonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6]. The method involves chromatography of purified RNA polymerase on single-stranded DNA-agarose, Bio-Rex 70, and finally Ultragel AcA44. The sigma factor obtained is electrophoretically pure with a yield of about 40%. A number of the chemical--physical properties of sigma are presented. A molecular weight of 82,000 was determined by phosphate buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Ultraviolet absorption spectra were used to determine an E280nm 1% of 8.4. The amino acid composition and 12-residue N-terminal sequence (Met-Glx-Glx-Asx-Pro-Glx-(Ser or Cys)-Glx-Leu-Lys-Leu-Leu) of sigma have been determined. The isoelectric focusing properties of sigma are presented. Denaturation--renaturation studies indicate that sigma is capable of an unusually rapid and complete recovery of activity after being subjected to denaturing conditions. A stable, 40,000-dalton fragment is generated from sigma by mild trypsin treatment.     

Regulation of RNA polymerase subunit synthesis in Escherichia coli: utilization of DNA-Intercalating drugs as a probe.

The effect of four DNA-intercalating drugs on the synthesis of the β and β′ subunits of Escherichia coli RNA polymerase was investigated. Acridine orange, proflavine, ethidium bromide, and berberine sulfate at sublethal doses caused a general reduction in cellular RNA and protein syntheses. Under this condition, acridine orange and proflavine rapidly led to overproduction of the β and β′ subunits in significant amounts. Ethidium bromide and berberine sulfate also caused overproduction of the two subunits but with a delay of 10 min at 30 °C. The β and β′ subunits of RNA polymerase became the major proteins being synthesized by E. coli cells after prolonged treatment with DNA-intercalating drugs. The level of the α subunit of RNA polymerase was not altered by any of the drugs tested. The overproduction of the β and β′ subunits induced by DNA-intercalating drugs is shown to require de novo synthesis of the ββ′ mRNA. These findings indicate that the expression of the ββ′ operon is regulated and that the synthesis of the α subunit is not regulated by the mechanism regulating the ββ′ operon. Taken together with evidence reported by others, these results strongly suggest that the concentration of intracellular free RNA polymerase plays a role in regulating the expression of the ββ′ operon.     

Escherichia coli integration host factor inhibits the NusA stimulation of RNA polymerase sigma subunit synthesis in vitro

As reported previously, Integration Host Factor (IHF) stimulates cII expression but the stimulatory effect is prevented by the NusA protein (Peacock and Weissbach, 1985, Biochem. Biophys. Res. Commun. 127, 1026-1031). The interaction between IHF and the NusA protein has been investigated further in studies on the in vitro expression of the genes for the beta (rpoB) and sigma (rpoD) subunits of RNA polymerase, both known to be stimulated by NusA. The NusA stimulation of rpoD expression can be prevented by IHF, but IHF has no effect by itself on rpoD expression. IHF does not influence rpoB expression either in the presence or absence of NusA.     

A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity

A mutation is described that alters the promoter specificity of sigma 70, the primary sigma factor of Escherichia coli RNA polymerase. In strains carrying both the mutant and wild-type sigma gene (rpoD), the mutant sigma causes a large increase in the activity of mutant P22 ant promoters with A.T or C.G instead of the wild-type, consensus G.C base-pair at position -33, the third position of the consensus -35 hexamer 5'-TTGACA-3'. There is little or no effect on the activities of the wild-type and 23 other mutant ant promoters, including one with T.A at -33. The mutant sigma also activates E. coli lac promoters with A.T or C.G, but not T.A, at the corresponding position. The rpoD mutation (rpoD-RH588) changes a CGT codon to CAT. The corresponding change in sigma 70 is Arg588----His. This residue is in a region that is conserved among most sigma factors, a region that is also homologous with the helix-turn-helix motif of DNA-binding proteins. These results suggest that this region of sigma 70 is directly involved in recognition of the -35 hexamer.     

RNA polymerase mutant with altered sigma factor in Escherichia coli.

Summary E. gracilis chloroplast DNA Bam fragments E and D, coding for rRNA were cloned separately using the plasmid pBR 322 as vector and E. coli as host. The newly constructed recombinant plasmids EgcKS 8 and EgcKS 11 (containing the Bam HI fragments E and D respectively) were analysed and characterized by gel electrophoresis, electronmicroscopy and analytical ultracentrifugation.Abbreviations Ap Ampicillin - Tc Tetracycline-hydrochloride - Bam HI endonuclease isolated from Bacillus amyloliquefaciens - Eco RI endonuclease isolated from E. coli RY13 - Bgl II endonuclease isolated from Bacillus globiggi - EDTA Ethylene-diamine-tetra-acetic-acid - ctDNA chloroplast DNA An abstract of this work was presented at the 10th annual meeting of the Union Schweizerischer Gesellschaften für Experimentelle Biologie, Davos 19th and 20th Mai, 1978. The recommendations of the Schweizerische Akademie für medizinische Wissenschaften for work with recombinant DNA-molecules were respected throughout this work.     

Subunits of RNA polymerase in function and structure. IV. Enhancing role of sigma in the subunit assembly of Escherichia coli RNA polymerase

The in vitro reconstitution of DNA-dependent RNA polymerase of Escherichia coli is markedly enhanced by the σ subunit. This conclusion is based on the following observations: (1) the core activity was higher for the enzyme reconstituted from mixtures of α, β,β′ and σ subunits than from those devoid of the σ subunit; (2) the reconstituted enzyme lacking the σ subunit could never regain full activity even when the σ subunit was supplied before assay and (3) the recovery of enzyme activity increased in proportion to the amount of σ subunit present during reconstitution.This influence of the σ subunit was also observed when reconstitution was carried out by mixing the α₂β complex and the β′ subunit, the second step in the sequence of enzyme formation. The σ subunit-dependent assembly between the α₂β complex and the β′ subunit required an ionic strength of around 0.2 m-KC1 and was enhanced by higher temperatures. In contrast, formation of the α₂β complex, which exhibited no requirement for the σ subunit, was unaffected by the salt concentration used or the temperature of reaction. The enhancement was observed not only at neutral but also at alkaline pH. The native enzyme per se was greatly activated after brief exposure to alkali.     

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.     

Regulation of Escherichia coli sigma F RNA polymerase by flhD and flhC flagellar regulatory genes.

The sigma F RNA polymerase has been characterized biochemically and is known to transcribe several flagellar genes in Escherichia coli. It was found that while the flagellar regulatory genes flhD and flhC are required for sigma F activity, the sizes of their corresponding gene products are inconsistent with their encoding sigma F itself.