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.     

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.     

Non-coordinate regulation of enzymes involved in transfer RNA metabolism in Escherichia coli

During different steady state growth conditions in Escherichia coli the level of the three tRNA-modifying enzymes, the tRNA(m5Urd)-, tRNA(m1Guo)- and tRNA(mam5s2Urd)methyltransferase and of five aminoacyl-tRNA synthetases, the leucyl-, valyl-, isoleucyl-, arginyl- and threonyl-tRNA-synthetase, has been determined. It is shown that those two classes of tRNA affecting enzymes are not coordinately regulated and that even within these two groups of enzymes the constituents are regulated independently of each other. Furthermore it is demonstrated that none of the aminoacyl-tRNA synthetases and only one of the three tRNA-methyltransferases, the tRNA(m5Urd)methyltransferase, is under control of the relA+-gene.     

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 DNAAn 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.     

Inducible DNA polymerase I synthesis in a UV hyper-resistant mutant of Escherichia coli

A mutant of Escherichia coli which is more resistant to shortwave UV light than its wild-type parent strain and which can synthesise DNA polymerase I constitutively has been further analysed. It carries two mutational alleles which are located about 1.5 min apart and cotransducible by P1 with the argH locus. The two mutational alleles have been segregated and their analysis shows that one of them is responsible for UV hyper-resistance whereas the other mutation confers UV sensitivity. Recombinant plasmids carrying various sections of the polA regulatory region, linked to a galK gene, were introduced into the mutant strains. Analysis of galactokinase shows that the enzyme activity in the UV hyper-resistant mutant is increased. The results suggest that the synthesis of DNA polymerase I in E. coli is inducible.     

Gene expression in a temperature-sensitive gyrB mutant of Escherichia coli.

The effect of gyrase inactivation on gene expression was studied by examining the activities of different promoters in a temperature-sensitive gyrB mutant of Escherichia coli. The relative activities of promoters affected by cAMP-binding protein (CAP), e.g., the lac promoter, are not reduced by gyrase inactivation but can, on the contrary, be enhanced. This stimulation depends on the promoter location or its structure. The tnaA promoter is activated when located near the origin of replication, suggesting a differential effect of gyrase inactivation on various chromosomal domains. Only silent or mutant promoters such as the non-functional wild-type bgl or the lacIq can be activated. No differential effect of gyrase inactivation on the lambda pL and the trp promoters carried by the phage can be detected.