Subunits of RNA polymerase in function and structure: VI. Sequence of the assembly in vitro of Escherichia coli RNA polymerase

Detailed analysis of the assembly in vitro of Escherichia coli RNA polymerase reveals that core enzyme subunits are assembled in the following sequence: $\text{2 α → α}{}_2\text{→}\text{β}\text{α}{}_2\text{β}\text{→}\text{β′}\text{α}{}_2\text{ββ′}$(premature core) → E (active core). Activation of the premature core enzyme, the rate-limiting step in this sequence, can be achieved in three different ways: self-reactivation, sigma subunit (σ or σ′)-promoted reactivation, and DNA-promoted reactivation.Although there has been disagreement on the enhancement of core enzyme maturation by sigma subunit or DNA, the discrepancy is resolved by the present finding that the premature core alone can be activated in the presence of high concentrations of salt or glycerol, whereas at a salt concentration as low as that in vivo, sigma subunit or DNA is required for maximum activation. However, the question remains unsolved as to which of the three ways operates in the in vivo process of RNA polymerase formation.     

Subunits of RNA polymerase in function and structure. 3. Accumulation of the intermediate alpha 2beta in the subunit assembly of Escherichia coli RNA polymerase by treatment with cyanate

The reactivation of DNA-dependent RNA polymerase, dissociated with 6 M-urea, was markedly reduced when aged solutions of urea were used without deionization. Cyanate, which is in equilibrium with urea in aged solutions, had similar effects on the reversible dissociation of the RNA polymerase suggesting that the inhibitor in aged urea solutions might be cyanate. The presence of dithiothreitol and MgCl₂ in the dissociation buffer prevented this inhibition and, moreover, these agents could activate inactive subunits. Thus, it is suggested that the inhibition might be due to carbamylation of cysteine residues of the subunits of the polymerase.Upon exposure to cyanate, the β′ subunit was preferentially inactivated; as a result, the α₂β complex, an intermediate in the sequence of the in vitro assembly of the RNA polymerase, accumulated when reconstitution was carried out with cyanate-treated subunits. This finding further strengthened the proposal that the subunits of this multimeric enzyme assemble in the following sequence: 2α + β + β′ → α₂β + β′ → α₂ββ′.     

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 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.     

Identification of a subunit assembly domain in the alpha subunit of Escherichia coli RNA polymerase

The alpha subunit of Escherichia coli DNA-dependent RNA polymerase is encoded by the rpoA gene and plays a major role in enzyme assembly. A set of C-terminal deletion mutations of the rpoA gene was constructed. The results of mixed reconstitution experiments in vitro, using the truncated alpha polypeptides encoded by the rpoA deletion mutants, suggest that the amino-terminal two-thirds of alpha subunit is sufficient for the formation of pseudo-core complexes containing both beta and beta' subunits.     

Subunits of RNA polymerase in function and structure; Maturation in vitro of core enzyme from Escherichia coli

Reconstitution of Escherichia coli RNA polymerase was found to be markedly enhanced by DNA as well as by the σ subunit. Among discrete steps of subunit assembly, formation of the primary intermediate α₂β complex and subsequent association of the complex with the β′ subunit are not affected by the presence of DNA and the σ subunit; the α₂ββ′ complex thus formed, however, is virtually inactive and is subject to temperature-dependent activation by DNA and the σ subunit. The α₂ββ′ complex is, therefore, a secondary intermediate in the sequence of enzyme formation, or a premature form of core enzyme.In the course of activation of the premature core complex, the subunit σ interacts with both the α₂β complex and the β′ subunit; DNA acts in much the same way. The enzyme, reconstituted in the presence of DNA, is recovered attached to the DNA, added as an enhancer, and initiates RNA synthesis without prior release from the DNA. A limited number of unique DNA sites appear to be concerned with the enzyme maturation.     

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.     

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)