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)     

Biosynthesis of RNA polymerase in Escherichia coli

Summary In spite of the generally well-coordinated synthesis of RNA polymerase core enzyme subunits (, and ) in Escherichia coli, a situation was found during the growth transition from exponential to stationary phase in which this coordination was broken (the order of differential repression being ; Kawakami et al. (1979)). The present study indicates that, during a certain period of the growth transition, twice as much subunit is synthesized as subunit and the overproduced subunit accumulates as the assembly intermediate ₂ complex, which is rapidly and preferentially degraded.Two independent factors, i.e., carbon source down-shift and oxygen depletion, were examined separately for their influence on the coordinated regulation of the synthesis of RNA polymerase subunits. The depletion of glucose added as a sole carbon source was accompanied by repression of the synthesis of all core enzyme subunits, while under the same conditions the differential rate of subunit synthesis increased. In contrast, the sudden ending of the oxygen supply resulted in specific repression of the synthesis of only and subunits but not of and subunits. The latter result may be explained by the autogenous repression of the rpoBC genes by a temporal increase in the amount of unused cytoplasmic RNA polymerase.Paper XI in this series is Kawakami and Ishihama (1980)     

On the role of the Escherichia coli RNA polymerase sigma factor in T4 phage development

Summary The rpoD800 mutation causing the temperature sensitivity of Escherichia coli RNA polymerase sigma factor has been used to demonstrate that the bacterial sigma factor is necessary throughout T4 phage development. In T4-infected RpoD800 mutant cells RNA synthesis is equally depressed at restrictive temperature at early and late stages of infection. The results show the bacterial sigma factor to be necessary for the synthesis of all RNA types in infected cells.     

Restriction map of the hepatitis B virus DNA cloned in Escherichia coli

A plasmid carrying the complete genome of hepatitis B virus (HBV) inserted into the BamHI site of the pBR322 plasmid vector has been constructed. The physical map of HBV DNA established for 13 restriction endonucleases allows to conclude that the cloned DNA is similar, but not identical to the HBV DNA of ayw subtype.     

Amber mutations in the structural gene for RNA polymerase sigma factor of Escherichia coli

Summary Amber mutants of Escherichia coli K-12 affected in the structural gene (rpoD) for th subunit of RNA polymerase have been obtained from a strain harboring a temperature-sensitive amber suppressor (supF-Ts6) which is active only at low temperatures. These mutants grow normally at low temperature (30°C) but do not grow at high temperature (42°C) due to the inability to synthesize factor. In one mutant studied in detail (rpoD40), the rate of -factor synthesis at 30°C is about half that of the wild type and is decreased to 10%–15% within 1 h of incubation at 42°C. The synthesis of core polymerase subunits or bulk protein is virtually unaffected at least for 2 h. The defect of the mutant in synthesis and growth at high temperature can be suppressed by any of the amber suppressors tested (supD, supE or supF). RNA-polymerase holoenzymes prepared from the mutant cells carrying each of the suppressors (grown at 42°C) exhibit different thermostabilities attributable to alterations in the factor. The reduced synthesis in the mutant is accompanied by the synthesis of polypeptide tentatively identified as amber fragment. These results as well as the genetic mapping data indicate that the amber mutation (rpoD40) resides within the structural gene for the factor and directly affects synthesis upon inactivation of the suppressor at high temperature.     

Escherichia coli RNA polymerase binding to a DNA terminus prevents formation of a closed promoter complex

A 302 bp DNA fragment and a 113 bp subfragment of the former, both containing the fd gene VIII promoter (P VIII), were found to exhibit temperature-dependent differential behaviour in RNA chain initiation from P VIII. At 37°C no significant differences were observed, while at 17°C chain initiation was strongly suppressed only with the 113 bp fragment. This phenomenon depended on the presence of the (blunt) DNA terminus upstream from P VIII (position −70). Footprinting revealed that at 17°C RNA polymerase was bound to this DNA fragment in a different mode. Contacts were observed only upstream from position −25. On the contrary, at 37°C only the promoter complex footprint was visible. These results indicate that at 17°C formation of the non-initiating complex is more favourable than formation of the promoter complex (which is closed at 17°C; Hofer, B., Müller, D. and Köster, H. (1985) Nucleic Acids Res. 13, 5995–6013) and that formation of both complexes is mutually exclusive. No footprints of RNA polymerase were observed at other DNA termini. This indicates a sequence-specificity for the interaction at the terminus of the 113 bp fragment. The footprint pattern, together with features of the DNA sequence, suggests that the contacts involved in this interaction are similar to those promoter contacts formed upstream from position −20 and that DNA without a −10 region can be specifically recognized by RNA polymerase.     

Escherichia coli mutants temperature-sensitive for DNA synthesis

Summary A series of mutants of E. coli temperature-sensitive for DNA synthesis has been studied. The temperature-sensitive DNA mutations map in seven distinct genetic loci most of which have not been previously reported. Mutations in dnaA and in dnaC affect the initiation of DNA replication; those at the remaining loci affect chain elongation. A temperature-sensitive Flac is shown to suppress a group A mutant with somewhat less efficiency than other F factors previously reported by others. The gene products rendered temperaturesensitive by the mutations have not been identified for any of the loci.     

Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45°C in the absence and presence of Mg²⁺. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. ΔH⁰ and ΔS⁰ for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, ΔH⁰ ≈ 15–16 kcal/mol (63–67 kJ/mol) and ΔS⁰ ≈ 50–57 cal/K per mol (209–239 J/K per mol). The presence of Mg²⁺ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg²⁺, the ΔH⁰ and ΔS⁰ values are larger and temperature-dependent, with ΔH⁰ ≈ 22 kcal/mol (92 kJ/mol) and ΔS⁰ ≈ 72 cal/K per mol (approx. 300 J/K per mol) at 25°C, and ΔC_p⁰ 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.     

Deletion map of the Escherichia coli K-12 dnaB gene

Summary Acid phosphatase isoenzymes of Chlamydomonas reinhardii were investigated by isoelectric focusing in polyacrylamide gel systems. In this paper we describe in detail an original method for isoelectric focusing of acid phosphatases extracted from wildtype and acid phosphatase-lacking mutant algae, obtained from Laboratoire de Génetique of University of Liège. Three isoenzymes can be separated from the buffer-soluble components of these cells. An additional isoenzyme type can be visualized using the nonionic detergent NP40 as solubilizer. We conclude that these four isoenzymes are releated to the structural gene of the soluble constitutive acid phosphatase, which was shown by their appearance in P ₂ and their total absence in mutant P _a. The pl values of soluble constitutive acid phosphatase isoenzymes range between pH 5.2 and 6.2. As a result of treatment with NP40 the extracts from both wild-type and mutant lines contain two additional active phosphatase forms which can be characterized by their high heat resistance and low pI values. These enzymes are fully active using either -naphthyl phosphate or different acetate esters as substrates.     

Inverted duplication in the genome of the temperate Streptomyces phage SH3

Summary The DNA of the temperate Streptomyces phage SH3 contains 100 base-pair long inverted repeats separated by a 940 base-pair long segment of DNA as revealed by electromicroscopic analysis of snapback structures formed after rapid intrastrand reannealing of denatured DNA. The inverted repeat structure was found preferentially at map unit 22 of the circular physical map, in rare cases also in other positions, suggesting a movable character of this genetic element.     

Escherichia coli merodiploids with altered ratios of episomal to chromosomal RNA polymerase activity

Summary Starting with an E. coli merodiploid strain (rpoB ⁺ (rifS)/rpoB^- (rifR)) containing equal amounts of rifampicin-sensitive and resistant RNA polymerase activities, mutants were isolated that had a disproportionately high ratio of the rifampicin-resistant enzyme activity. With one strain it is shown that the mutation responsible for this phenotype is closely linked to the rifR (rpoB^-) allele.