Changes in the concentrations of guanosine 5'-diphosphate 3'-diphosphate and the initiating nucleoside triphosphate account for inhibition of rRNA transcription in fructose-1,6-diphosphate aldolase (fda) mutants

Abstract

Early screens for conditional lethal mutations that affected rRNA expression in Escherichia coli identified temperature-sensitive fda mutants (fda encodes the glycolytic enzyme fructose-1,6-diphosphate aldolase). It was shown that these fda(Ts) mutants were severely impaired in rRNA synthesis upon shift to the restrictive temperature, although the mechanism of inhibition was never determined. Here, we bring resolution to this long-standing question by showing that changes in the concentrations of guanosine 5'-diphosphate 3'-diphosphate and initiating nucleoside triphosphates can account for the previously observed effects of fda mutations on rRNA transcription.

FIG. 1.

Effects of the fda(Ts) mutation on transcription and on ppGpp and iNTP concentrations. rrnB P1 and rrnB P1(dis) promoter activities were measured by primer extension (13) in wild-type (A) and ts8 (B) strains before and as a function of time after a shift from 30 to 42°C. The promoters contained rrnB P1 DNA sequences from −66 to + 9 with respect to the transcription start site (+1) and synthesized an unstable RNA (18). Cultures (rrnB P1, RLG6256; rrnB P1(dis), RLG6257; rrnB P1 fda, RLG6258; rrnB P1(dis) fda, RLG6259) were grown in morpholinepropanesulfonic acid (MOPS) minimal medium (15) supplemented with 0.4% glucose, 0.4% Casamino Acids, 40 μg of Trp/ml, 5 μg of thiamine/ml, 1 mM KH₂PO₄, and 100 μg of ampicillin/ml. The asterisk in panel B indicates that promoter activity at this time point was at or below background. ATP and iNTP concentrations were extracted with formic acid and measured by thin-layer chromatography as described previously (18) from wild-type (C) and ts8 (D) strains grown in parallel with the cultures in panels A and B, except 20 μCi of KH₂³²PO₄/ml was added to the medium. Values were normalized to those at time zero (just before the temperature shift). Each point represents the average of at least two measurements from independent cultures. Error is indicated.

FIG. 2.

Effects of fda(Ts) on promoter activity and ATP concentrations in ΔrelA ΔspoT strains. rrnB P1 and rrnB P1(dis) activities were measured by primer extension in ΔrelA ΔspoT (A) and ΔrelA ΔspoT (B) fda strains as detailed in the legend to Fig. 1. Strains described in the legend to Fig. 1 were sequentially transduced to ΔrelA ΔspoT as described previously (18) and were grown as indicated in the legend, except that the medium contained 1.0% Casamino Acids. ATP concentrations were measured from ΔrelA ΔspoT (C) and ΔrelA ΔspoT fda (D) strains grown as indicated in the legend for panels A and B except 20 μCi of KH₂³²PO₄/ml was included in the medium. Each point represents the average of at least two measurements from independent cultures. Error is indicated.