Transcriptional Responses to ppGpp and DksA

Abstract

The stringent response to nutrient deprivation is a stress response found throughout the bacterial domain of life. Although first described in proteobacteria for matching ribosome synthesis to the cell's translation status and for preventing formation of defective ribosomal particles, the response is actually much broader, regulating many hundreds of genes-some positively, some negatively. Utilization of the signaling molecules ppGpp and pppGpp for this purpose is ubiquitous in bacterial evolution, although the mechanisms employed vary. In proteobacteria, the signaling molecules typically bind to two sites on RNA polymerase, one at the interface of the β' and ω subunits and one at the interface of the β' secondary channel and the transcription factor DksA. The β' secondary channel is targeted by other transcription regulators as well. Although studies on the transcriptional outputs of the stringent response date back at least 50 years, the mechanisms responsible are only now coming into focus.

Keywords: DksA; TraR; ppGpp; promoter; stress response; stringent response.

Figure 1

Structural models of transcription factors DksA (151 residues) and TraR (73 residues) adapted from PDB1TJL (92) and PDB5W1S (82), respectively. Each protein contains a zinc ion bound in the globular domain and a C-terminal helix (CTH). The N terminus of each protein is indicated. Residues shown in red interact with RNA polymerase (RNAP) near the active site and are required for function but not for binding to RNAP (Asp74, Ala76 in the coiled-coil tip of DksA; Asp6, Ala8 at the N-terminal end of TraR) (45, 70). Residues shown in blue in DksA (Leu95, Lys98, Arg129, Lys139) form part of ppGpp Binding Site 2 but are not required for DksA binding to RNAP (99). Residues in blue in TraR (Ile20, Glu66) are required for function but not for binding to RNAP (45).

Figure 2

Transcription in vitro was performed with ppGpp and/or DksA and with Escherichia coli RNA polymerase (RNAP) containing or lacking the ω subunit, as indicated. The resulting RNAPs thereby contain the ppGpp binding sites indicated below the bars. Reactions were performed on supercoiled plasmid DNA templates containing (a) the rrnB P1 promoter or (b) the iraP promoter. The ppGpp concentration was 200 µM, and the DksA concentration was 2 µM. Transcription with RNAP lacking both ppGpp binding sites is set at 1.0 (data are from Reference 99).

Figure 3

Surface representation of Escherichia coli RNAP showing locations of ppGpp Binding Site 1 (ω-β′ interface) and Binding Site 2 (DksA-β′ interface). Adapted in Pymol from PDB4JKR and PDB1TJL. Site 1 position as in PDB4JKR; Site 2 position modeled based on genetic/biochemical data as described Reference . RNAP subunits, RNAP β SI1, secondary channel, and active site are indicated. Abbreviations: NTD, N-terminal domain; RNAP, RNA polymerase.

Figure 4

Three paradigms for regulation of transcription initiation by ppGpp: (a) In Escherichia coli, ppGpp (red star) binds directly to two sites on RNA polymerase (RNAP, gray oval). DksA is represented by a blue triangle (99). (b) In Bacillus subtilis, ppGpp binds to enzymes responsible for GTP biosynthesis (e.g., GMK, HPRT, blue circles), indirectly inhibiting transcription from rRNA operons by reducing the concentration of the initiating nucleotide GTP (64, 77). (c) In Francisella tularensis, ppGpp binds to the RNAP-associated heterodimer MglA/SspA (dark blue circles), leading to its interaction with the transcriptional activator PigR (blue oval) (25, 28).

Figure 5

Distribution of TraR homologs in bacteria and bacteriophages. TraR-like proteins were identified by NCBI Blast (https://www.ncbi.nlm.nih.gov/blast), and a phylogenetic tree depicting their distribution was constructed using phyloT (http://phylot.biobyte.de/). Only representative species are shown. Red font is used for phyla or families in which there are proteins approximately the same length as TraR with key conserved residues. Blue font is used for phyla in which TraR-like proteins were not identified. A list of species with TraR homologs is in Reference .