Characterization of RNA synthesis in an Escherichia coli mutant with a temperature-sensitive lesion in stable RNA synthesis

Previous experiments with Escherichia coli strain 2S142 have shown that the synthesis of stable RNA is preferentially blocked at the restrictive temperature. In this paper, we have examined the capacity of this mutant strain to synthesize RNA in vitro. Growth of the strain for as short a period as 10 min at 42 degrees C resulted in a 40 to 60% loss of RNA synthetic capacity and a fourfold decrease in percent rRNA synthesized in toluenized cell preparations. The time course for the loss and recovery of this RNA synthetic capacity correlated very well with the changes in RNA synthesis observed in vivo. We found no difference in temperature sensitivity of the purified RNA polymerase from the mutant and the parental strains. Moreover, there was no detectable alteration in the amount of enzyme, specific activity of the enzyme, or electrophoretic mobility of the subunits when the mutant strain was grown at 42 degrees C. The capacity for rRNA synthesis was also measured with the Zubay in vitro system (Reiness et al., Proc. Natl. Acad. Sci. 72:2881-2885, 1975). Supernatant fractions (S-30) prepared from cells grown at 30 degrees C were capable of up to 31.2% rRNA synthesis, using phi 80d3 DNA as template. S-30 fractions from cells grown at 42 degrees C synthesized 8.6% rRNA. The bottom one-third of the S-100 fraction and the ribosomal salt wash from 30 degrees C cells contained one or more factors which partially restored preferential rRNA synthesis in S-30 fractions from cells grown at 42 degrees C. Preliminary evidence suggests that the factor(s) is protein in nature.     

Biosynthesis of the beta and beta' subunits of RNA polymerase in Escherichia coli

The amounts of the β and β′ subunits of the DNA-dependent RNA polymerase relative to the amount of total protein synthesized have been determined under a number of growth conditions in two strains of Escherichia coli. The results of these measurements have been expressed as the relative rate of synthesis of core RNA polymerase, α_p, assuming the four constituent subunits (2α, 1β and 1β′) to be synthesized in equivalent amounts.This quantity, α_p, was found not to vary greatly with the growth rate μ. For glucose-grown cells of E. coli B/r (μ = 1.5 doublings/h) α_p = 1.4%, corresponding to about 7000 molecules of core RNA polymerase per cell. For slowgrowing cells the value obtained for α_p is lower and for fast-growing cells somewhat 3 higher. The comparison of these values with the number of RNA polymerase molecules estimated to be actively engaged in RNA synthesis indicates that both slow- and fast-growing cells contain a surplus of RNA polymerase, if the catalytic unit is assumed to be the monomer of core RNA polymerase.In addition to the measurements of cells during balanced growth at various rates, α_p has been determined during the transition from one growth rate to another and during synchronous growth. During a shift-up the rate of synthesis of polymerase follows closely the rate of total protein synthesis, α_p being nearly constant for a period of twenty minutes after the shift. In a synchronously dividing culture of E. coli B/r, α_p was seen to be fairly constant during two cycles of synchronous division. It appears that α_p is rather insensitive to the effect of gene doubling during the cell cycle.     

A temperature-sensitive mutant of Escherichia coli with an altered RNA polymerase beta' subunit.

$\text{Penicillium charlesii}$ extracts contain UDP-galactose:NAD⁺ 2-hexosyl oxidoreductase (1). ADP-ribose also serves as a substrate resulting in formation of NADH and an oxidized ADP-ribose derivative. Treatment of the oxidized product with NaBH₄ followed by hydrolysis at pH 2 and 100° releases xylose as well as ribose. We conclude that ADP-D-$\text{glycero}$-D-$\text{glycero}$-3-pentosulose (ADP-3-ketoribose) is the product derived from ADP-ribose.     

An Escherichia coli mutant exhibiting temperature-sensitive ATP synthesis

A mutant strain SM434 (ttr-3) of Escherichia coli that exhibits a temperature-sensitive Unc(succinate-nonutilizing) phenotype was characterized. The mutant allele ttr-3 was not linked to the ilvA gene, but was complemented by Fill carrying 81 min-91 min of the E. coli chromosome. The mutant strain SM434 exhibited resistance to N,N'-dicyclohexylcarbodiimide (DCCD) and a temperature-sensitive phenotype at the level of ATP synthesis, compatible with that of cell growth. These findings indicate that the mutant strain SM434 could carry a mutation (ttr-3) in an unknown gene responsible for the energy-transduction system.     

An amber mutation in the gene encoding the beta'' subunit of Escherichia coli RNA polymerase.

An Escherichia coli strain carrying an amber mutation (UAG) in rpoC, the gene encoding the beta prime subunit of RNA polymerase, was isolated after mutagenesis with nitrosoguanidine. The mutation was moved into an unmutagenized strain carrying the supD43,74 allele, which encodes a temperature-sensitive su1 amber suppressor, and sue alleles, which enhance the efficiency of the suppressor. In this background, beta prime is not synthesized at high temperature. Suppression of the mutation by the non-temperature-sensitive amber suppressor su1+ yields a protein which is functional at all temperatures examined (30, 37, and 42 degrees C).     

Alteration of RNA I metabolism in a temperature-sensitive Escherichia coli rnpA mutant strain.

E. coli strain A49 carries the themosensitive mutation in the rnpA gene encoding the protein component of RNase P, a tRNA-processing enzyme. Two small RNAs were highly accumulated in the A49 carrying derivatives of ColE1-type plasmids, at nonpermissive temperature. Characterization of these RNAs showed that they were the processed or degraded products derived from RNA I, which is the negative controller of ColE1-type plasmid replication. These derivatives of RNA I only differ in size at the 5' ends. The data of their degradation and synthesis kinetics suggest that they are intermediates of RNA I metabolism.     

Cold sensitive mutant of Escherichia coli with altered RNA polymerase

Summary Cold sensitive (cs) mutants of E. coli unable to grow at 26° C were isolated. Three of about 200 mutants tested have RNA polymerase which is about 5 times less active at low temperatures than the wild type RNA polymerase. One of such mutants cs 1 was studied in more detail. The mutation affects core enzyme and is closely linked to rif-r mutations. By transduction analysis the following order of markers was established: argH — rif-r — cs 1.     

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.