Direct regulation of Escherichia coli ribosomal protein promoters by the transcription factors ppGpp and DksA

Abstract

We show here that the promoters for many of the Escherichia coli ribosomal protein operons are regulated directly by two transcription factors, the small RNA polymerase-binding protein DksA and the nutritional stress-induced nucleotide ppGpp. ppGpp and DksA work together to inhibit transcription initiation from ribosomal protein promoters in vitro and in vivo. The degree of promoter regulation by ppGpp/DksA varies among the r-protein promoters, but some are inhibited almost as much as rRNA promoters. Thus, many r-protein operons are regulated at the level of transcription in addition to their control by the classic translational feedback systems discovered ~30 y ago. We conclude that direct control of r-protein promoters and rRNA promoters by the same signal, ppGpp/DksA, makes a major contribution to the balanced and coordinated synthesis rates of all of the ribosomal components.

Fig. 1.

Effects of ppGpp/DksA on transcription from r-protein promoters in vitro and in vivo. (A) Single-round transcription in vitro. Bars represent transcription in the presence of DksA and ppGpp relative to that in the absence of either factor. Error bars represent the SD from at least six reactions. Promoters are generally identified by the first gene in the operon; alternative names are given in parentheses. (B) Expression from promoter-lacZ fusions, log phase (OD₆₀₀ ~0.3). Bars represent β-galactosidase activity in the WT strain relative to that in the ΔdksA strain. Absolute activities varied slightly from day to day, but the WT:ΔdksA ratio remained constant. Reported error bars represent means and SDs from WT: ΔdksA ratios in multiple experiments. (C) Expression from promoter-lacZ fusions, stationary phase. β-Galactosidase assays were performed on aliquots from the same cultures used in B but 22–24 h after inoculation.

Fig. 2.

Stringent control of r-protein promoters in WT and ΔdksA mutant strains. The promoters tested in A–G are listed in the lower left of each panel. Transcription was from the same promoter–lacZ fusion constructs used in Fig. 1 B and C, but quantified from direct measurement of RNA by primer extension at different times after addition of serine hydroxamate (SHX) to starve for amino acids (Materials and Methods). Activities are plotted relative to that from the same culture before addition of SHX. Error bars represent range from two independent experiments.

Fig. 3.

Independent and combined effects of ppGpp and DksA on r-protein promoters in vitro. The promoters tested in A–G are listed at the top of each panel. Promoter activities were measured in the presence of DksA, ppGpp, both, or neither as indicated under the bottom panels using single-round in vitro transcription assays (Materials and Methods). Representative duplicate lanes of relevant bands from a single gel are shown above the bars in the histograms, but error bars indicate the SD from at least six reactions. The amount of transcription was normalized to that in the absence of either factor.

Fig. 4.

Ribosomal protein promoter complexes require similar DksA concentrations for transcription inhibition. Half-maximal concentration of DksA required for transcription inhibition in vitro (IC₅₀) was measured at a range of DksA concentrations in the presence of 100 μM ppGpp. The r-protein promoter corresponding to the colored titration curves in A are identified in B, along with the IC₅₀ for each promoter and the SE.

Fig. 5.

Schematic diagram illustrating transcriptional and translational contributions to regulation of ribosome synthesis in E. coli. Many r-protein promoters and all rRNA promoters are directly regulated by ppGpp/DksA. In addition, r-protein expression is translationally regulated by binding of excess repressor r-proteins to r-protein mRNAs.